Sensors employing combinatorial artificial receptors

ABSTRACT

The present invention relates to sensors and sensor systems that utilize combinational artificial receptors. Embodiments of the present invention employ combinational artificial receptors in electromagnetic (e.g. optical) and electrochemical sensors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/934,193, filed Sep. 3, 2004, which claims priority to U.S.Provisional Patent Application Nos. 60/499,752, 60/500,081, 60/499,776,60/499,867, 60/499,965, and 60/499,975 each filed Sep. 3, 2003; and60/526,511 60/526,699, 60/526,703, 60/526,708, and 60/527,190 each filedDec. 2, 2003. Each of these patent applications is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to sensors employing artificial receptors,such as combinatorial artificial receptor arrays. The present receptorsinclude heterogeneous and immobilized combinations of building blockmolecules. In certain embodiments, combinations of 2, 3, 4, or 5distinct building block molecules immobilized near one another on asupport provide molecular structures that can be employed in sensorsystems. Sensors employing the present artificial receptors can detectthe receptor's ligand.

BACKGROUND

The preparation of artificial receptors that bind ligands like proteins,peptides, carbohydrates, microbes, pollutants, pharmaceuticals, and thelike with high sensitivity and specificity is an active area ofresearch. None of the conventional approaches has been particularlysuccessful; achieving only modest sensitivity and specificity mainly dueto low binding affinity.

Antibodies, enzymes, and natural receptors generally have bindingconstants in the 10⁸-10¹² range, which results in both nanomolarsensitivity and targeted specificity. By contrast, conventionalartificial receptors typically have binding constants of about 10³ to10⁵, with the predictable result of millimolar sensitivity and limitedspecificity.

Several conventional approaches are being pursued in attempts to achievehighly sensitive and specific artificial receptors. These approachesinclude, for example, affinity isolation, molecular imprinting, andrational and/or combinatorial design and synthesis of synthetic orsemi-synthetic receptors.

Such rational or combinatorial approaches have been limited by therelatively small number of receptors which are evaluated and/or by theirreliance on a design strategy which focuses on only one building block,the homogeneous design strategy. Common combinatorial approaches formmicroarrays that include 10,000 or 100,000 distinct spots on a standardmicroscope slide. However, such conventional methods for combinatorialsynthesis provide a single molecule per spot. Employing a singlebuilding block in each spot provides only a single possible receptor perspot. Synthesis of thousands of building blocks would be required tomake thousands of possible receptors.

Further, these conventional approaches are hampered by the currentlylimited understanding of the principals which lead to efficient bindingand the large number of possible structures for receptors, which makessuch an approach problematic.

There remains a need for methods for detecting ligands and for detectingcompounds that disrupt one or more binding interactions.

SUMMARY

An embodiment of a sensor system includes a waveguide, a detectionsystem that can be operatively coupled to the waveguide, and a workingartificial receptor. The waveguide can be operatively configured withrespect to the working artificial receptor such that the waveguide iscapable of receiving light that from the viscininty of the workingartificial receptor. The detection system can be configured to detectlight from the waveguide.

An embodiment of an electrochemical sensing system includes a workingelectrode, a reference electrode, and a working artificial receptor thatis coupled to the working electrode. The sensing system can beconfigured to generate a sensing signal.

An embodiment of an electrochemical sensing system includes a fieldeffect transistor, and a working artificial receptor that is coupled tothe field effect transistor. A signal can be generated by the fieldeffect transistor when a test ligand binds to a working artificialreceptor.

An embodiment of sensor system includes a detector and a workingartificial receptor that is coupled to the detector. The detector systemcan be configured to detect the presence of a test ligand bound to theworking artificial receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates two dimensional representations of anembodiment of a receptor according to the present invention that employs4 different building blocks to make a ligand binding site.

FIG. 2 schematically illustrates two and three dimensionalrepresentations of an embodiment of a molecular configuration of 4building blocks, each building block including a recognition element, aframework, and a linker coupled to a support (immobilization/anchor).

FIG. 3 schematically illustrates an embodiment of the present methodsand artificial receptors employing shuffling and exchanging buildingblocks.

FIG. 4 is a schematic drawing of an electromagnetic radiation (light)sensor system that includes a wave guide.

FIG. 5 is a schematic drawing of an optical sensor system that includesa plurality of fibers.

FIG. 6 is a schematic drawing of an electrochemical sensor system.

FIG. 7 is a schematic drawing of an electrochemical sensor system thatemploys a membrane to which the present artificial receptors can becoupled.

FIG. 8 is a schematic drawing of a sensor system that uses both opticaland electrochemical sensors.

FIG. 9 is a cross-sectional view of a microwell and a protrusion.

FIG. 10 is an exemplary illustration of a pattern detected by a sensorarray.

FIG. 11 is a schematic illustration of a fiber bundle to which thepresent artificial receptors can be coupled.

FIGS. 12A-12D are schematic illustrations of field effect transistordevices to which the present artificial receptors can be bound.

FIG. 13 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand,such as a molecule or cell.

FIG. 14 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors.

FIG. 15 schematically illustrates certain binding patterns on an arrayof working artificial receptors.

FIG. 16 schematically illustrates an embodiment of a method fordeveloping a method and system for detecting a test ligand.

FIG. 17 schematically illustrates an embodiment of a method fordetecting an agent that disrupts a binding interaction of a targetmolecule.

FIG. 18 schematically illustrates an embodiment of a method fordetecting an agent that disrupts a binding interaction of a complexincluding a target molecule.

FIG. 19 schematically illustrates an embodiment of a method of employingthe present artificial receptors to produce or as an affinity support.

FIG. 20 schematically illustrates a candidate disruptor disrupting aprotein:protein complex.

FIG. 21 schematically illustrates evaluating an array of candidateartificial receptors for binding of a test ligand and selecting one ormore working artificial receptors for binding or operating on a testligand.

FIG. 22 schematically illustrates identification of a lead artificialreceptor from among candidate artificial receptors.

FIG. 23 schematically illustrates a false color fluorescence image of alabeled microarray according to an embodiment of the present invention.

FIG. 24 schematically illustrates a two dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding phycoerythrin.

FIG. 25 schematically illustrates a three dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding phycoerythrin.

FIG. 26 schematically illustrates a two dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a fluorescent derivative of ovalbumin.

FIG. 27 schematically illustrates a three dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a fluorescent derivative of ovalbumin.

FIG. 28 schematically illustrates a two dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a fluorescent derivative of bovine serum albumin.

FIG. 29 schematically illustrates a three dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a fluorescent derivative of bovine serum albumin.

FIG. 30 schematically illustrates a two dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding an acetylated horseradish peroxidase.

FIG. 31 schematically illustrates a three dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding an acetylated horseradish peroxidase.

FIG. 32 schematically illustrates a two dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a TCDD derivative of horseradish peroxidase.

FIG. 33 schematically illustrates a three dimensional plot of dataobtained for candidate artificial receptors contacted with and/orbinding a TCDD derivative of horseradish peroxidase.

FIG. 34 schematically illustrates a subset of the data illustrated inFIG. 25.

FIG. 35 schematically illustrates a subset of the data illustrated inFIG. 25.

FIG. 36 schematically illustrates a subset of the data illustrated inFIG. 25.

FIG. 37 schematically illustrates a correlation of binding data forphycoerythrin against logP for the building blocks making up theartificial receptor.

FIG. 38 schematically illustrates a correlation of binding data forphycoerythrin against logP for the building blocks making up theartificial receptor.

FIG. 39 schematically illustrates a two dimensional plot comparing dataobtained for candidate artificial receptors contacted with and/orbinding phycoerythrin to data obtained for candidate artificialreceptors contacted with and/or binding a fluorescent derivative ofbovine serum albumin.

FIGS. 40, 41, and 42 schematically illustrate subsets of data from FIGS.25, 29, and 27, respectively, and demonstrate that the array ofartificial receptors according to the present invention yields receptorsdistinguished between three analytes, phycoerythrin, bovine serumalbumin, and ovalbumin.

FIG. 43 schematically illustrates a gray scale image of the fluorescencesignal from a scan of a control plate which was prepared by washing offthe building blocks with organic solvent before incubation with the testligand.

FIG. 44 schematically illustrates a gray scale image of the fluorescencesignal from a scan of an experimental plate which was incubated with 1.0μg/ml Cholera Toxin B at 23° C.

FIG. 45 schematically illustrates a gray scale image of the fluorescencesignal from a scan of an experimental plate which was incubated with 1.0μg/ml Cholera Toxin B at 3° C.

FIG. 46 schematically illustrates a gray scale image of the fluorescencesignal from a scan of an experimental plate which was incubated with 1.0μg/ml Cholera Toxin B at 43° C.

FIGS. 47-49 schematically illustrate plots of the fluorescence signalsobtained from the candidate artificial receptors illustrated in FIGS.44-46.

FIG. 50 schematically illustrate plots of the fluorescence signalsobtained from the combinations of building blocks employed in thepresent studies, when those building blocks are covalently linked to thesupport. Binding was conducted at 23° C.

FIG. 51 schematically illustrates a graph of the changes in fluorescencesignal from individual combinations of building blocks at 4° C., 23° C.,or 44° C.

FIG. 52 schematically illustrates a graph of the changes in fluorescencesignal from individual combinations of building blocks at 4° C., 23° C.,or 44° C.

FIG. 53 schematically illustrates the data presented in FIG. 51 (linesmarked A) and the data presented in FIG. 52 (lines marked B).

FIG. 54 schematically illustrates a graph of the fluorescence signal at44° C. divided by the signal at 23° C. against the fluorescence signalobtained from binding at 23° C. for the artificial receptors withreversibly immobilized receptors.

FIG. 55 illustrates fluorescence signals produced by binding of choleratoxin to a microarray of the present candidate artificial receptorsfollowed by washing with buffer in an experiment reported in Example 4.

FIG. 56 illustrates the fluorescence signals due to cholera toxinbinding that were detected upon competition with GM1 OS (0.34 μM) in anexperiment reported in Example 4.

FIG. 57 illustrates the ratio of the amount bound in the absence of GM1OS to the amount bound in competition with GM1 OS (0.34 μM) in anexperiment reported in Example 4.

FIG. 58 illustrates fluorescence signals produced by binding of choleratoxin to a microarray of the present candidate artificial receptorsfollowed by washing with buffer in an experiment reported in Example 4and for comparison with competition experiments using 5.1 μM GM1 OS.

FIG. 59 illustrates the fluorescence signals due to cholera toxinbinding that were detected upon competition with GM1 OS (5.1 μM) in anexperiment reported in Example 4.

FIG. 60 illustrates the ratio of the amount bound in the absence of GM1OS to the amount bound in competition with GM1 OS (5.1 μM) in anexperiment reported in Example 4.

FIG. 61 illustrates the fluorescence signals produced by binding ofcholera toxin to the microarray of candidate artificial receptors aloneand in competition with each of the three concentrations of GM1 in theexperiment reported in Example 5.

FIG. 62 illustrates the ratio of the amount bound in the absence of GM1OS to the amount bound upon competition with GM1 for the lowconcentration of GM1 employed in Example 5.

FIG. 63 illustrates the fluorescence signals produced by binding ofcholera toxin to the microarray of candidate artificial receptorswithout pretreatment with GM1 in the experiment reported in Example 6.

FIGS. 64-66 illustrate the fluorescence signals produced by binding ofcholera toxin to the microarray of candidate artificial receptors withpretreatment with GM1 (100 μg/ml, 10 μg/ml, and 1 μg/ml GM1,respectively) in the experiment reported in Example 6.

FIG. 67 illustrates the ratio of the amount bound in the presence of 1μg/ml GM1 to the amount bound in the absence of GM1 in the experimentreported in Example 6.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

As used herein, the term “peptide” refers to a compound including two ormore amino acid residues joined by amide bond(s).

As used herein, the terms “polypeptide” and “protein” refer to a peptideincluding more than about 20 amino acid residues connected by peptidelinkages.

As used herein, the term “proteome” refers to the expression profile ofthe proteins of an organism, tissue, organ, or cell. The proteome can bespecific to a particular status (e.g., development, health, etc.) of theorganism, tissue, organ, or cell.

As used herein, the term “support” refers to a solid support that is,typically, macroscopic.

As used herein, the term scaffold refers to a molecular scale structureto which a plurality of building blocks can covalently bind.

Reversibly immobilizing building blocks on a support couples thebuilding blocks to the support through a mechanism that allows thebuilding blocks to be uncoupled from the support without destroying orunacceptably degrading the building block or the support. That is,immobilization can be reversed without destroying or unacceptablydegrading the building block or the support. In an embodiment,immobilization can be reversed with only negligible or ineffectivelevels of degradation of the building block or the support. Reversibleimmobilization can employ readily reversible covalent bonding ornoncovalent interactions. Suitable noncovalent interactions includeinteractions between ions, hydrogen bonding, van der Waals interactions,and the like. Readily reversible covalent bonding refers to covalentbonds that can be formed and broken under conditions that do not destroyor unacceptably degrade the building block or the support.

A combination of building blocks immobilized on, for example, a supportcan be a candidate artificial receptor, a lead artificial receptor, or aworking artificial receptor. That is, a heterogeneous building blockspot on a slide or a plurality of building blocks coated on a tube orwell can be a candidate artificial receptor, a lead artificial receptor,or a working artificial receptor. A candidate artificial receptor canbecome a lead artificial receptor, which can become a working artificialreceptor.

As used herein the phrase “candidate artificial receptor” refers to animmobilized combination of building blocks that can be tested todetermine whether or not a particular test ligand binds to thatcombination. In an embodiment, the combination includes one or morereversibly immobilized building blocks. In an embodiment, the candidateartificial receptor can be a heterogeneous building block spot on aslide or a plurality of building blocks coated on a tube or well.

As used herein the phrase “lead artificial receptor” refers to animmobilized combination of building blocks that binds a test ligand at apredetermined concentration of test ligand, for example at 10, 1, 0.1,or 0.01 μg/ml, or at 1, 0.1, or 0.01 ng/ml. In an embodiment, thecombination includes one or more reversibly immobilized building blocks.In an embodiment, the lead artificial receptor can be a heterogeneousbuilding block spot on a slide or a plurality of building blocks coatedon a tube or well.

As used herein the phrase “working artificial receptor” refers to acombination of building blocks that binds a test ligand with aselectivity and/or sensitivity effective for categorizing or identifyingthe test ligand. That is, binding to that combination of building blocksdescribes the test ligand as belonging to a category of test ligands oras being a particular test ligand. A working artificial receptor can,for example, bind the ligand at a concentration of, for example, 100,10, 1, 0.1, 0.01, or 0.001 ng/ml. In an embodiment, the combinationincludes one or more reversibly immobilized building blocks. In anembodiment, the working artificial receptor can be a heterogeneousbuilding block spot on a slide or a plurality of building blocks coatedon a tube, well, slide, or other support or on a scaffold.

As used herein the phrase “working artificial receptor complex” refersto a plurality of artificial receptors, each a combination of buildingblocks, that binds a test ligand with a pattern of selectivity and/orsensitivity effective for categorizing or identifying the test ligand.That is, binding to the several receptors of the complex describes thetest ligand as belonging to a category of test ligands or as being aparticular test ligand. The individual receptors in the complex can eachbind the ligand at different concentrations or with differentaffinities. For example, the individual receptors in the complex eachbind the ligand at concentrations of 100, 10, 1, 0.1, 0.01 or 0.001ng/ml. In an embodiment, the combination includes one or more reversiblyimmobilized building blocks. In an embodiment, the working artificialreceptor complex can be a plurality of heterogeneous building blockspots or regions on a slide; a plurality of wells, each coated with adifferent combination of building blocks; or a plurality of tubes, eachcoated with a different combination of building blocks.

As used herein, the phrase “significant number of candidate artificialreceptors” refers to sufficient candidate artificial receptors toprovide an opportunity to find a working artificial receptor, workingartificial receptor complex, or lead artificial receptor. As few asabout 100 to about 200 candidate artificial receptors can be asignificant number for finding working artificial receptor complexessuitable for distinguishing two proteins (e.g., cholera toxin andphycoerythrin). In other embodiments, a significant number of candidateartificial receptors can include about 1,000 candidate artificialreceptors, about 10,000 candidate artificial receptors, about 100,000candidate artificial receptors, or more.

Although not limiting to the present invention, it is believed that thesignificant number of candidate artificial receptors required to providean opportunity to find a working artificial receptor may be larger thanthe significant number required to find a working artificial receptorcomplex. Although not limiting to the present invention, it is believedthat the significant number of candidate artificial receptors requiredto provide an opportunity to find a lead artificial receptor may belarger than the significant number required to find a working artificialreceptor. Although not limiting to the present invention, it is believedthat the significant number of candidate artificial receptors requiredto provide an opportunity to find a working artificial receptor for atest ligand with few features may be more than for a test ligand withmany features.

As used herein, the term “building block” refers to a molecularcomponent of an artificial receptor including portions that can beenvisioned as or that include one or more linkers, one or moreframeworks, and one or more recognition elements. In an embodiment, thebuilding block includes a linker, a framework, and one or morerecognition elements. In an embodiment, the linker includes a moietysuitable for reversibly immobilizing the building block, for example, ona support, surface or lawn. The building block interacts with theligand.

As used herein, the term “linker” refers to a portion of or functionalgroup on a building block that can be employed to or that does (e.g.,reversibly) couple the building block to a support, for example, throughcovalent link, ionic interaction, electrostatic interaction, orhydrophobic interaction.

As used herein, the term “framework” refers to a portion of a buildingblock including the linker or to which the linker is coupled and towhich one or more recognition elements are coupled.

As used herein, the term “recognition element” refers to a portion of abuilding block coupled to the framework but not covalently coupled tothe support. Although not limiting to the present invention, therecognition element can provide or form one or more groups, surfaces, orspaces for interacting with the ligand.

As used herein, the phrase “plurality of building blocks” refers to twoor more building blocks of different structure in a mixture, in a kit,or on a support or scaffold. Each building block has a particularstructure, and use of building blocks in the plural, or of a pluralityof building blocks, refers to more than one of these particularstructures. Building blocks or plurality of building blocks does notrefer to a plurality of molecules each having the same structure.

As used herein, the phrase “combination of building blocks” refers to aplurality of building blocks that together are in a spot, region, or acandidate, lead, or working artificial receptor. A combination ofbuilding blocks can be a subset of a set of building blocks. Forexample, a combination of building blocks can be one of the possiblecombinations of 2, 3, 4, 5, or 6 building blocks from a set of N (e.g.,N=10-200) building blocks.

As used herein, the phrases “homogenous immobilized building block” and“homogenous immobilized building blocks” refer to a support or spothaving immobilized on or within it only a single building block.

As used herein, the phrase “activated building block” refers to abuilding block activated to make it ready to form a covalent bond to afunctional group, for example, on a support. A building block includinga carboxyl group can be converted to a building block including anactivated ester group, which is an activated building block. Anactivated building block including an activated ester group can react,for example, with an amine to form a covalent bond.

As used herein, the term “naïve” used with respect to one or morebuilding blocks refers to a building block that has not previously beendetermined or known to bind to a test ligand of interest. For example,the recognition element(s) on a naïve building block has not previouslybeen determined or known to bind to a test ligand of interest. Abuilding block that is or includes a known ligand (e.g., GM1) for aparticular protein (test ligand) of interest (e.g., cholera toxin) isnot naïve with respect to that protein (test ligand).

As used herein, the term “immobilized” used with respect to buildingblocks coupled to a support refers to building blocks being stablyoriented on the support so that they do not migrate on the support orrelease from the support. Building blocks can be immobilized by covalentcoupling, by ionic interactions, by electrostatic interactions, such asion pairing, or by hydrophobic interactions, such as van der Waalsinteractions.

As used herein a “region” of a support, tube, well, or surface refers toa contiguous portion of the support, tube, well, or surface. Buildingblocks coupled to a region can refer to building blocks in proximity toone another in that region.

As used herein, a “bulky” group on a molecule is larger than a moietyincluding 7 or 8 carbon atoms.

As used herein, a “small” group on a molecule is hydrogen, methyl, oranother group smaller than a moiety including 4 carbon atoms.

As used herein, the term “lawn” refers to a layer, spot, or region offunctional groups on a support, for example, at a density sufficient toplace coupled building blocks in proximity to one another. Thefunctional groups can include groups capable of forming covalent, ionic,electrostatic, or hydrophobic interactions with building blocks.

As used herein, the term “alkyl” refers to saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₁₂ for straight chain, C₁-C₆ for branched chain).Likewise, cycloalkyls can have from 3-10 carbon atoms in their ringstructure, for example, 5, 6 or 7 carbons in the ring structure.

The term “alkyl” as used herein refers to both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, a formyl,or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aryl alkyl, or an aromaticor heteroaromatic moiety. The moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For example, thesubstituents of a substituted alkyl can include substituted andunsubstituted forms of the groups listed above.

The phrase “aryl alkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

As used herein, the terms “alkenyl” and “alkynyl” refer to unsaturatedaliphatic groups analogous in length and optional substitution to thealkyls groups described above, but that contain at least one double ortriple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents such as those described above foralkyl groups. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings (the rings are “fused rings”) wherein at leastone of the rings is aromatic, e.g., the other cyclic ring(s) can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

As used herein, the terms “heterocycle” or “heterocyclic group” refer to3- to 12-membered ring structures, e.g., 3- to 7-membered rings, whosering structures include one to four heteroatoms. Heterocyclyl groupsinclude, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents such as those described for alkyl groups.

As used herein, the term “heteroatom” as used herein means an atom ofany element other than carbon or hydrogen, such as nitrogen, oxygen,sulfur and phosphorous.

Overview of the Artificial Receptor

FIG. 1 schematically illustrates an embodiment employing 4 distinctbuilding blocks in a spot on a microarray to make a ligand binding site.This Figure illustrates a group of 4 building blocks at the corners of asquare forming a unit cell. A group of four building blocks can beenvisioned as the vertices on any quadrilateral. FIG. 1 illustrates thatspots or regions of building blocks can be envisioned as multiple unitcells, in this illustration square unit cells. Groups of unit cells offour building blocks in the shape of other quadrilaterals can also beformed on a support.

Each immobilized building block molecule can provide one or more “arms”extending from a “framework” and each can include groups that interactwith a ligand or with portions of another immobilized building block.FIG. 2 illustrates that combinations of four building blocks, eachincluding a framework with two arms (called “recognition elements”),provides a molecular configuration of building blocks that form a sitefor binding a ligand. Such a site formed by building blocks such asthose exemplified below can bind a small molecule, such as a drug,metabolite, pollutant, or the like, and/or can bind a larger ligand suchas a macromolecule or microbe.

The present artificial receptors can include building blocks reversiblyimmobilized on a support or surface. Reversing immobilization of thebuilding blocks can allow movement of building blocks to a differentlocation on the support or surface, or exchange of building blocks ontoand off of the surface. For example, the combinations of building blockscan bind a ligand when reversibly coupled to or immobilized on thesupport. Reversing the coupling or immobilization of the building blocksprovides opportunity for rearranging the building blocks, which canimprove binding of the ligand. Further, the present invention can allowfor adding additional or different building blocks, which can furtherimprove binding of a ligand.

FIG. 3 schematically illustrates an embodiment employing an initialartificial receptor surface (A) with four different building blocks onthe surface, which are represented by shaded shapes. This initialartificial receptor surface (A) undergoes (1) binding of a ligand to anartificial receptor and (2) shuffling the building blocks on thereceptor surface to yield a lead artificial receptor (B). Shufflingrefers to reversing the coupling or immobilization of the buildingblocks and allowing their rearrangement on the receptor surface. Afterforming a lead artificial receptor, additional building blocks can be(3) exchanged onto and/or off of the receptor surface (C). Exchangingrefers to building blocks leaving the surface and entering a solutioncontacting the surface and/or building blocks leaving a solutioncontacting the surface and becoming part of the artificial receptor. Theadditional building blocks can be selected for structural diversity(e.g., randomly) or selected based on the structure of the buildingblocks in the lead artificial receptor to provide additional avenues forimproving binding. The original and additional building blocks can thenbe (4) shuffled and exchanged to provide higher affinity artificialreceptors on the surface (D).

Sensors Employing the Artificial Receptors

The present artificial receptors can be configured in sensors, sensorsystems, and sensing methods. For example, a sensor system can be can beconfigured to detect a test ligand in a sample or an environment basedupon a signal received from a sensor operatively configured to detect atest ligand that binds to the present artificial receptor.

In an embodiment, the sensor system includes an optical sensor system.The optical sensor system can include a waveguide, a detection systemoperatively coupled to the waveguide, and an artificial receptor thatbinds a test ligand of interest (e.g., a working artificial receptor).The waveguide can be operatively configured with respect to the workingartificial receptor such that the waveguide can receive an opticalsignal (e.g., fluorescence, luminescence, or absorbance) from theworking artificial receptor.

In an embodiment, the sensor system includes an electrochemical sensorsystem. An electrochemical sensing system can include a transducer(e.g., an electrode or CHEMFET), a detection system operatively coupledto the transducer, and an artificial receptor that binds a test ligandof interest (e.g., a working artificial receptor). The transducer can beoperatively configured with respect to the working artificial receptorsuch that the transducer can detect changes in electrical charge,potential, or current (e.g., conductance, capacitance, or impedence)from the working artificial receptor. In an embodiment, the transducerincludes at least one electrode. An electrochemical sensing system caninclude a working electrode, a reference electrode, and a workingartificial receptor. In an embodiment, the present artificial receptorscan be coupled to the working electrode. In an embodiment, the presentartificial receptors can be coupled to a membrane that is configuredbetween the working electrode and the reference electrode. In anembodiment, the working electrode and reference electrode can beconventional electrodes. In an embodiment, the working electrode andreference electrode can be a source and a drain of a field effecttransistor.

A sample expected of containing a test ligand can be brought intocontact with an artificial receptor by a variety of mechanisms. Forexample, the sample can be carried in a stream of air or an aerosol. Thesample can be a liquid. In an embodiment, it can be desirable toincrease the surface area to which present artificial receptors arecoupled to provide enhanced detection of a test ligand in a gaseous orliquid flow. In an embodiment, a flow can be received through a foamedsupport substance, such as an aerogel to which present artificialreceptors can be coupled.

Supporting Environments for the Artificial Receptors

The present artificial receptors can be supported in sensors or sensorssystems in a variety of configurations in or on a variety of supportmaterials. For example, the present artificial receptors can besupported in solid, gel, and foam environments. To accommodate varioussensing systems, the present artificial receptors can be configured inan environment that conducts electrical currents, light, and/or otherelectromagnetic radiation.

In an embodiment, present artificial receptors can be located at or neara distal surface of a fiber, such an optical fiber. The distal surfacecan, for example, be an end of an optical fiber. In an embodiment, thepresent artificial receptors can be bonded or otherwise coupled to thedistal surface on the fiber. For example, the present artificialreceptors can be coupled directly to the end of a fiber. In anembodiment, the present artificial receptors can be coupled, embedded,or otherwise configured to a support material or substrate that can becoupled to the fiber. In an embodiment, the present artificial receptorscan be located near the end of a fiber without being coupled to thefiber. For example, the present artificial receptors can be coupled toor suspended in a target object or substrate that can be configured tobe detectable by the fiber. In an embodiment, a receptor-supportingmaterial can be layered on the object or substrate. In an embodiment,the target object itself can be the support for the receptor, i.e. theobject or substrate can be formed from the material to which the presentartificial receptors can be coupled.

In an embodiment, a sol-gel can be a support for the present artificialreceptors. For example, the present artificial receptors can be coupledto or suspended in a sol-gel that can be coupled to a fiber orelectrode. The sol-gel process involves the transition of a system froma liquid “sol” into a solid “gel” phase. Through a sol-gel process, itis possible to fabricate ceramic or glass materials in a wide variety offorms. Dopants can be added to alter the glass properties. For example,ultra-fine or spherical shaped powders, thin film coatings, ceramicfibers, microporous inorganic membranes, monolithic ceramics andglasses, or extremely porous aerogel materials can be developed using asol gel process. In one process, a sol gel is a colloidal suspension ofsilica particles that is gelled to form a solid. The resulting porousgel can be chemically purified and consolidated at high temperaturesinto high purity silica.

A sol-gel can be coated or otherwise applied to an object, oralternatively formed into an object itself. In an embodiment, a sol-gelcan be applied to the surface of a fiber by dip coating, and the currentartificial receptors can be coupled to the sol gel. In an embodiment,the present artificial receptors can be coupled to a sol gel substratethat is configured to be detectable by a fiber but is not connected tothe fiber. For example, sol gel can be applied to a plate or othersubstrate, and the optical fiber can be aligned to detect a portion ofthe plate.

In addition to dip-coating, sol-gel can be applied through otherprocesses such as spraying, casting, and spin coating. Other forms ofsol gels, such as aerogels, powders, and thin films formed from xerogelscan also be configured for use with the present artificial receptors.Sol gel processes are described in U.S. Pat. No. 5,774,603. Dual-layersensors and sensors that utilize aerosol-generated sol gels aredescribed in U.S. Pat. No. 6,016,689.

In an embodiment, a microsphere (or “bead”) can be configured as asupport for the present artificial receptors. A sensor incorporating anoptical fiber and a solid porous inorganic microsphere is described inU.S. Pat. No. 5,496,997. Microspheres having a polymeric shell withconsistent shell thickness are described in U.S. Pat. No. 6,720,007.Microspheres are also discussed in U.S. Pat. Nos. 6,327,410 and6,266,459 and Published U.S. Patent Application Nos. 20030016897,20020122612, 20010029049. A plurality or multiplicity of microspherescan be configured to allow for multiplexed detection (binding ofmultiple analytes on multiple sets of coated beads). The presentartificial receptors can also be incorporated into a nanosphere.

In an embodiment, a support, such as a microsphere can be encoded withan optical signature. While other support environments and structurescan be encoded, microspheres will be referenced herein for descriptivepurposes. A structure such as a microsphere can be encoded, for example,by use of a dye that can be entrapped within the bead. The dye can, forexample, be a fluorescent dye. The dye can also be a chromophore orphosphor, or other optically-detectable substance. In an embodiment, twoor more dyes can be used to provide a multi-parametric code. In anembodiment, the code can correspond to a chemical functionality orsensitivity that is associated with the microsphere. Fiber optic sensorswith encoded microspheres are described in U.S. Pat. No. 6,023,540.

In an embodiment, the present artificial receptors can be configured foruse in conjunction with a capillary tube. For example, an optical fiber(or an electrode) can be supported within a capillary tube. A fluidsample can be introduced into the space between the fiber (or electrode)and the tube. A fluid can be drawn into and supported in the space bycapillary action. The present artificial receptors can be coupled to theoptical fiber (or electrode), the capillary tube, or an object in thecapillary tube, and configured to detect a test ligand in the fluidsample. A fluorescent immunoassay employing an optical fiber in acapillary tube is described in U.S. Pat. No. 4,447,546.

The present artificial receptors can also be configured in a cavity. Asused herein, cavity is intended to refer to a microwell, pit, cavity,depression, void, or similar structure. For purposes of the presentdiscussion, a microwell will be described, although the presentapplication also applies to other forms of cavities. In an embodiment, amicrowell can be formed in an end of an optical fiber. A microwell canalso be formed on a substrate that can be configured to be detectable byan optical fiber. Microwell-based sensors are described in U.S. Pat.Nos. 6,667,159, 6,377,721, and 6,210,910. In an embodiment, a pluralityof microwells can be formed at the distal end of individual fiberswithin a randomly-ordered addressable sensor arrays, as furtherdescribed in U.S. Pat. No. 6,667,159.

In an embodiment, a microwell receives a bead, to which receptors can becoupled. In an embodiment, the microwell and bead can be configured onan end surface of a fiber. In an embodiment, the bead and depressionstructure can mimic the shape of mammalian taste buds. The bead can, forexample, be constructed of a polymer.

FIG. 9 shows a cross-section of a depression 730 in a substance 720which can be, for example, a substrate, waveguide, or electrode. Thedepression can be, for example, a microwell. A protrusion 740, which canbe a sphere or bead, can reside in the depression 730. The presentworking electrodes 710 can be coupled to the protrusion 740. In anembodiment, the present working electrodes can be coupled to thedepression surface.

In an embodiment, an array of micron-sized wells can be formed at thedistal tip of an optical imaging fiber, and the present artificialreceptors can be coupled in the wells. In an embodiment, a well can becreated using a wet-etch process. Where there are differences in coreand clad etch rates, a well can be created between core and cladmaterial. For example, where fiber cores etch faster than the cladding,the core etches away to produce a well that is defined by the remainingclad. In an embodiment, the present artificial receptors can be coupledto the cells. In an embodiment, a living cell can be received into awell and the present artificial receptors can be configured to detectthe presence and/or behavior of the living cells.

In an embodiment, cells can be plated onto a fiber and the presentartificial receptors can be configured to facilitate detection of thecells.

In an embodiment, sensors employing the present artificial receptors canbe configured to make optical analytical measurements at remotelocations. U.S. Pat. No. 5,814,524 describes an apparatus that employsan imaging fiber comprising a fiber optic array and a Gradient Indexlens and utilizes a remotely-positioned solid substrate having lightenergy absorbing indicator ligands on an external surface for reactivecontact with individual species of analytes when present in a fluidsample.

The present artificial receptors can also be supported in a carbonpaste. In an embodiment, a hybrid optical/electrochemical sensor can beprovided, where the carbon paste can be electrically coupled to acircuit that can be configured to detect electrical behaviors orproperties of the sample/and or receptors.

The present artificial receptors can also be configured in amicrochannel. In an embodiment, a microchannel includes a longitudinalrecess on a surface and the present artificial receptors can be coupledto the recessed surface and configured to detect a test ligand in afluid in the microchannel.

The present artificial receptors can also be supported on a membrane. Inan embodiment, the present artificial receptors can be coupled to amembrane which can be placed in front of the tip of an optical fiber. Inan embodiment, the membrane can be removable. In an embodiment, thepresent artificial receptor can be coupled to a membrane that ispositioned between two electrodes in an electrochemical sensor.

In an embodiment, the present artificial receptors can be coupled to ahexamethyldisiloxane plasma-polymerized film.

Other materials, including molecularly imprinted polymers and zeolite,can be configured as a support for the present artificial receptors. Thepresent artificial receptors can also be configured for use withdisposable screen printing technology.

Fiber Optic Sensors

Fiber optic technology can be utilized with the present artificialreceptors. Fiber optic systems can be configured to allow for detectionof modulation in the quality or quantity of electromagnetic radiation,such as visible light, infrared radiation, or ultraviolet radiation. Forexample, the intensity (energy), polarization state, phase andwavelength of radiation can be detected and measured. Embodiments offiber optic-based sensors are described in U.S. Pat. Nos. 5,690,894 and6,680,206.

A fiber optic detection system can be configured with a fiber arrangedto detect radiation and/or optical properties in the vicinity of thepresent artificial receptors. Embodiments of fiber optic sensing systemscan be designed to provide remote, distributed and multiplexed sensing.An embodiment of an optical sensing system that uses receptors is shownand described in U.S. Pat. No. 5,512,490. In an embodiment, sensors canbe employed with micromechanical systems. Sensors can also be designedto operate in hostile and hazardous environments. In an embodiment, afiber can be covered by a protective cladding layer to protect thefiber.

Fiber optic fibers can be interfaced with filters and light sources tofacilitate detection of particular properties or behaviors of asubstance that contains the present artificial receptors or is coupledto the present artificial receptors. The presence of a test ligandcoupled to a receptor in a sample can be detected, for example, bydetection of the emission, absorption, or refractive index of thesample. Detectors can be configured to measure a variety of types ofradiation, including, for example, white light, ultraviolet light orfluorescence. In an embodiment, an emitter and a receiver can be coupledto separate fibers and the reflected, refracted, or conducted light (orother radiation) can be detected. Other sources of radiation can also beused.

FIG. 4 is a schematic illustration of an embodiment of a sensor system200 that can be configured to detect an optical signal. The presentartificial receptors 210 can be coupled to a substrate 220. An lightsource 230, such as a laser or lamp, directs light toward the artificialreceptors. A waveguide 240 can be configured to detect electromagneticradiation, i.e. light. The waveguide 240 can be configured to detect,for example, reflected radiation, conducted radiation, or fluorescentradiation. The waveguide can be, for example, a planar waveguide. Afiber 250, for example an optical fiber, can be coupled to the waveguide240 and to an electromagnetic detection system 260, such as a chargecoupled device (CCD). The detection system can be coupled to aprocessing system 270, such as a computer system, which can beconfigured to process and save data received from the detection system.Alternatively, the detection system can be configured to be directlypresent or save data, without use of a separate processing system.

FIG. 5 shows an embodiment of a sensing system 300 in which a fiber 320can be configured for use as a waveguide. As shown in FIG. 5, multiplefibers can be configured together to detect light or other radiationreflected, refracted, conducted, or fluoresced by receptors 310. Datareceived from multiple fibers can be processed by a reception system 330to allow for observation of different locations on a substrate. In theembodiment shown in FIG. 5, the substrate 340 is not coupled to thefibers. In an alternative embodiment, a substrate can be coupled to adistal end of a fiber, and receptors can be coupled to the substrate. Inan embodiment, the present artificial receptors can be coupled directlyto the fiber.

Various types of optical sensors can be provided, such asintensity-based sensors, interference-based sensors, polarization-basedsensors, wavelength-based sensors, nonlinear optics-based sensors, andmultiphoton-based sensors. Other detectors such as photomultipliertubes, photo diodes, photodiode arrays, and microchannel plates can alsobe provided. Bundled fibers and fiber arrays can also be configured todetect a plurality of the present artificial sensors.

Fiber optic detection systems can operate based upon direct or indirectdetection. Directly detectable labels provide a directly detectablesignal without interaction with one or more additional chemical agents.Suitable directly detectable labels include colorimetric labels,fluorescent labels, and the like. Indirectly detectable labels interactwith one or more additional agents to provide a detectable signal.Suitable indirect labels include a labeled antibody and the like.

A detection system can be configured to detect the intrinsic opticalproperties of a sample, such as the refractive index, the color, or theemission (e.g. fluorescence) or absorption (e.g. quench fluorescence)properties of a sample. In an embodiment, the presence of a test ligandbound to a receptor can be detected based upon variations in one or moreproperties that are associated with the presence of the test ligand. Inan embodiment, the concentration of a test ligand or a label in a sensedregion is a function of the concentration of the analyte that binds to aparticular receptor, which can be determined from the optical propertiesor behavior of the sample. In an embodiment, a label can be bound to theanalyte. In an embodiment, a label can be bound to a blocking analyte.Use of labels is further described in U.S. Pat. No. 5,690,894.

Fiber optic systems can allow flexibility in detection configurations.For example, a fiber optic system can be configured to allow detectionin locations that can be otherwise difficult to access. Detection at adistance is also possible through use of one or more fibers. A group offibers can be configured to collect an array or data, as furtherdescribed below. Detection at multiple points along a fiber or a bundleof fibers is also possible.

A fiber optic detection system can be configured in a sensing or aprobing configuration. A sensing configuration allows for a stream ofdata. For example, a sensing system can be configured to sensecontinuously. A probing configuration takes a sample of data (i.e. a“snapshot”) at a particular point in time. A fiber optic probe can beconfigured to take a single data samples, or multiple samples. In anembodiment, for example, a fiber optic probe can be configured to takeperiodic data samples.

In an embodiment, the present artificial receptors can be configured inconjunction with an optical imaging fiber. An optical imaging fiber canbe formed from a multiplicity of individual fibers that are melted anddrawn together in a coherent manner such that an image can be carriedfrom one end of the fiber to the other. In an embodiment, an opticalimaging fiber can be configured to detect the presence, condition orbehavior of a ligand (e.g. a test ligand) that binds to the presentartificial receptors. Such imaging fibers can also be configured inconjunction with chemically sensitive polymer matrices to combineimaging and chemical sensing. This allows for simultaneous opticaldetection and measurement of chemical dynamics occurring in a sample.

In an embodiment, the present artificial receptors can be configured foruse in optical tweezers. An optical tweezer traps particles usingfocused laser beams to form optical traps. Optical tweezers aredescribed in published U.S. Patent Application No. 20030032204.

While fiber optic waveguides have been described in many of the aboveapplications, waveguides having other geometries (e.g. planar or strip)can also be configured for use with the receptors. Various modestructures (e.g. single-mode or multi-mode), refractive indexdistributions (step or gradient index) and material compositions (e.g.glass, polymer, semiconductor) can be employed. In an embodiment,receptors can be coupled to a surface of a planar wave guide. Signalscan be captured and conveyed through a fiber where they can be analyzedby a computer system. A fluorometric-based sensor system that uses aplanar waveguide is described in United States Published PatentApplication No. 2002/0160535.

Optical Signals

A detectable optical signal can be produced by the interaction of aligand with the present artificial receptor or through binding of alabeled moiety to the ligand. Fiber optic detection systems can beconfigured to detect a variety of optical signals, including thoseproduced by physical, chemical, or biological. For example, thedetection systems can be configured to detect one or more of refractiveindex, color, emission, absorption, fluorescence, or fluorescencequenching properties of a sample, and/or changes to those properties.

The presence of a test ligand binding to the present artificial receptorcan affect the quality or quantity of optical signal that is detectedthrough a fiber. The presence of two or more test ligands that are boundto the present artificial receptors (or to each other) can also generatedetectable changes in an optical signal or property.

In an embodiment, a change in fluorescence or quenching of fluorescencecan be detected. Fluorescence is the emission of radiation following theabsorption of radiation of a different wavelength. For example, anembodiment of a fluorescing substance can emit visible light after beingexposed to ultraviolet light. In an embodiment, the present artificialreceptor can include a fluorescent molecule whose fluorescent properties(e.g., emission intensity, emission wavelength, or lifetime) change uponanalyte binding. Sensors employing the present artificial receptors canbe configured to detect fluorescent decay time.

Sensors employing the present artificial receptors can be configured todetect the quenching of fluorescence. The quenching of fluorescence canoccur in a variety of forms. Dynamic quenching can occur where acollisional encounter between the quencher and the excited state isinvolved. The lifetime and intensity of the emission can be decreased bydynamic quenching. Concentration quenching can occur where a moleculequenches its own fluorescence at high concentration. ‘Static’ quenchingcan occur where an interaction between the fluorophore and quencher isinvolved. Color-quenching can occur where photons that are emitted arereabsorbed by a strongly colored component of the sample. Colorquenching is often accompanied by another quenching process based onFluorescent Resonance Energy Transfer (‘FRET’). This is a radiation-lessprocess where excited species transfer excitation energy to a neighborhaving an absorption that overlaps the fluorophore's emission spectrum.

Sensors configured with the present artificial receptors can also beconfigured to detect fluorescence polarization. Fluorescencepolarization refers to the fact that fluorescent molecules in solutionwhich are excited with plane-polarized light will emit light back into afixed plane if the molecules remain stationary during the excitation ofthe fluorophore. If the molecule rotates during the excited state, lightis emitted in a different plane. The intensity of light emitted can bemonitored in a plane to monitor the rotation of fluorescing molecules.Large molecules tend to rotate slower than small molecules. Fluorescencepolarization measurements can thus be used to study molecularinteractions. For example, the bound/free ratio of fluorescing moleculescan be detected. A sensor that detects the fluorescence polarization ofa label is described in U.S. Pat. No. 6,555,326.

Sensors employing the present artificial receptors can be configured todetect evanescent wave properties. Evanescent wave-based sensors aredescribed by U.S. Pat. Nos. 5,525,800, 5,639,668, and 6,731,827.

Sensors employing the present artificial receptors can be configured todetect phenomena such as chemiluminescence, bioluminescence, orchemibioluminescence. Chemiluminescence is the generation ofelectromagnetic radiation as light by the release of energy from achemical reaction. Chemical reactions using synthetic compounds andusually involving a highly oxidized species such as a peroxide arecommonly termed chemiluminescent reactions. Light-emitting reactionsarising from a living organism, such as the firefly or jellyfish, arecommonly termed bioluminescent reactions. Light-emitting reactions whichtake place by the use of electrical current are designatedelectrochemiluminescent reactions.

Sensors employing the present artificial receptors can be configured touse multi-parametric techniques. For example, multi-parametricfluorescence techniques can be performed based on based on spectralchange, intensity, lifetime and polarization of radiation. In anembodiment, for example, refractive index, emission, and abortionproperties can also be monitored concurrently. Other combinations arepossible.

In an embodiment, a charge-coupled device (CCD) camera can be configuredto detect optical signals from a system that incorporates the presentartificial receptors. In an embodiment, a long-duration exposure can beused to gather data over a period of time. Filters can also beconfigured to separate signals by wavelength.

In an embodiment, a near field array can be created on optical fibersand configured to observe an object to which the present artificialreceptor can be coupled.

Optical Sensor Arrays

In an embodiment, fiber optic sensors can be arranged to provide anarray of data. For example, in an embodiment, present artificialreceptors can be arranged on a surface in an array. The array of presentartificial receptors can be detected by a single fiber, or by a group offibers. In an embodiment, an array of present artificial receptors canbe positioned at the end of a fiber. For example, the present artificialreceptors can be coupled to the end of the fiber in an array pattern.Alternatively, present artificial receptors can be bonded, suspended, orotherwise positioned on an object, such as a glass plate, with a fiberoperatively arranged to allow detection with respect to the plate.

In an embodiment, an array of fibers can be configured with the presentartificial sensors to gather a data array. In an embodiment, an array offibers can be positioned to provide data samples from a variety ofpositions. In an embodiment, a variety of present artificial receptorswhich can be arranged in a pattern allow for simultaneous monitoring fora plurality of test ligands. Other variations or combinations of thesesensor techniques are possible. Optical fiber sensor arrays are furtherdescribed in U.S. Pat. Nos. 5,690,894, 6,667,159, and 6,680,206.

In an embodiment, a plurality of fibers can be configured in a bundle1100, as shown in FIG. 11. Particular receptors can be associated withparticular fibers, or groups of fibers. For example, in an embodiment,particular receptors can be coupled to fibers at a first end 1110 of abundle, and particular test ligands can be identified based upon signalsreceived at a second end 1120 of the bundle.

Techniques for analyzing a sample array include time-resolvedspectroscopy, spatially-resolved spectroscopy, evanescent wavespectroscopy, laser-assisted spectroscopy, surface plasmon spectroscopy,and multi-dimensional data acquisition. Fiber bundles can be configuredto form a sensor array, which can be used for imaging or for dataacquisition. In an embodiment, an artificial neural network can beconfigured to process data from a fiber bundle array. An artificialneural network deconvolutes a signal to allow association of data signalpatterns with the presence of a particular test ligand or group of testligands. For example, it may be determined that when a particular testligand is present, a particular combination of signals can be detectedthrough the fiber array. As data is collected, detection of a new testligand or combination of test ligands can be determined.

In an embodiment, sensors based on fiber optic technology in combinationwith the present artificial receptors can be configured to continuouslymeasure the concentration (or concentration changes) of variouscomponents of biological and environmental samples.

Electrochemical Sensors

The present artificial receptors can also be configured for use inelectrochemical sensors. The present artificial receptors can beconfigured for example with a variety of electrochemical sensors,including chemically modified electrode sensors and microelectrodes.Sensing schemes include, for example, voltametric and potentiometricmethods. Electrochemical sensors can be configured to detect any of avariety of test ligands.

In an embodiment, an electrochemical sensor includes the presentartificial receptors coupled to or near an electrode. In an embodiment,if a test ligand binds to a receptor, the presence of the test ligandcan be detected based on electrical monitoring. For example, thepresence of a particular test ligand can create an electrical charge.The electrical charge can create a detectable current or voltagedifferential. In an embodiment, when a chemically reactive gas is eitheroxidized (accepts oxygen and/or gives up electrons) or reduced (gives upoxygen and/or accepts electrons), a potential difference can be created,which can cause a current to flow. Non-reducible anions can be detectedbased upon electrochemical interaction between the anions and thereceptors or redox active moieties operatively coupled to the receptors.

In an embodiment, an electrochemical sensor includes a working electrode(or “sensing” electrode), a reference electrode, and the presentartificial receptor. The working electrode is exposed to a test ligand,and the reference electrode is not exposed to the test ligand. When thetest ligand is present at the working electrode, an electrochemicalreaction can occur. Typically, either an oxidation or reduction occurs,depending on the type of test ligand, but other reactions are possible.An oxidation reaction results in the flow of electrons from the workingelectrode to the reference electrode through an external circuit. Areduction reaction results in flow of electrons from the referenceelectrode to the working electrode. This flow of electrons in theelectric current is proportional to the test ligand concentration.

In an embodiment, present artificial receptors can be coupled to aworking electrode. The receptors can be exposed to a sample suspected ofcontaining a test ligand of interest. A chemical reaction can causeelectrons to flow to or from the working electrode. For example, in anembodiment, the receptors can be exposed to the sample, and a testligand in the sample can bind to the receptors. The receptors and boundtest ligand from the sample can be exposed to a reactive substance suchas an electrolyte, which can cause a chemical reaction, which in turncan cause a current to flow.

FIG. 6 shows an embodiment of an electrochemical sensor 400. The presentworking receptors 410 can be coupled to a working electrode 420. Workingelectrode 420 and a reference electrode 440 can be electrically coupledto an electrical sensing device 430, which can be configured to detect,for example, voltage or current. While receptors 410 are shown coupleddirectly to working electrode 420, the receptors can alternatively becoupled to a substrate that can be electrically coupled to the workingelectrode.

In an embodiment, an electrochemical sensor includes a membrane. Thepresent artificial receptors can be attached or otherwise coupled to themembrane. FIG. 7 shows an embodiment of an electrochemical sensor 500where the present working receptors 510 can be coupled to a membrane 520between a working electrode 530 and a reference electrode 540, which canbe coupled to an electrical sensing device 550. In an embodiment, themembrane is positioned between a working electrode and a test ligand.

In an embodiment, the present artificial receptors can be coupled to aconductive polymer film. For example, in an embodiment, the presentartificial receptors can be coupled to a conductive polymer film whichcan be coupled to the surface of a platinum electrode or otherelectrode. In an embodiment, the present artificial receptors can becoupled to a conducting polypyrrole film.

In an embodiment, a thick-film electrode can be integrated in or on aglass substrate. In an embodiment, a thin-film Ag/AgCl electrode can beintegrated on a glass substrate. Platinum electrodes can also beintegrated in or on the glass substrate. A multi-electrode system canalso be provided, with multiple electrodes being integrated in a singleglass substrate.

The present artificial receptors can also be coupled to asemiconductor-based sensor. In an embodiment, a semiconductor-basedsensor can be based on a electroadsorptive effect: An electrical fieldapplied on a sensitive layer of a semiconductor alters the adsorptioncharacteristics of the material.

The present artificial receptors can be configured with a field effecttransistor, such as a MOSFET, CHEMFET, ISFET, SAFET, or SGFET. Forexample, the present artificial receptors can be coupled the FET device,or to an object in an environment in the viscinity of a FET device. Inan embodiment, the present artificial receptors can be coupled to alayer of a metal-oxide-semiconductor (MOS) field effect transistor(FET), and the binding of a substance to an artificial receptor can bedetected through the MOSTFET. Schematic illustrations of a ISFET,ChemFET, SAFET, and SGFET are provided in FIGS. 12A-12D respectively.

In an embodiment, the present artificial receptors can be configuredwith an ion selective field effect transistor (ISFET). A schematicillustration of an embodiment of an ISFET device 1200 is shown in FIG.12A. In an embodiment, an ISFET operates based on effectsostatic effectscaused by a test ligand binding to the present artificial receptors. Inan embodiment, a chemically sensitive membrane 1205 or insulator layercan be configured to extend between a source 1210 and a drain 1215. Inan embodiment, the present artificial receptors 1220 can be coupled tothe chemically sensitive membrane or layer. Charges from chemicals thatcouple to the receptors and/or the membrane can be amplified through theoperation of the FET signal the presence and/or identity of a chemicalthat is bound to the receptors.

In an embodiment, the present artificial receptors can be coupled to achemically modified field effect transistor (ChemFET). A schematicillustration of a ChemFET 1225 is shown in FIG. 12B. In an embodiment,an oxide layer 1235 can extend between a drain 1245 and a source 1240,and a chemically sensitive gate 1250 can be layered on top of the oxidelayer. In an embodiment, the present artificial receptors can be coupledto or embedded in the oxide layer 1235 or the gate layer 1250. In anembodiment, the layer or layers can be a gel or a membrane. In anembodiment, both a gel and a membrane can be layered on a gate surface.

In an embodiment, the present artificial receptors can be coupled to asurface accessible field effect transisitor (SAFET). FIG. 12C shows aschematic illustration of a SAFET device 1255 that includes a source1275 and a drain 1280. In an embodiment, the present artificialreceptors 1260 can be coupled to an insulator structure 1265 or a gate1270.

In an embodiment, the present artificial receptors can be coupled to asuspended gate field effect transisitor (SGFET). FIG. 12D shows aschematic illustration of a SGFET 1285. In an embodiment, a chemicallysensitive insulator structure 1290 can be layered on a drain 1295 and asource 1300, and a gate 1305 can be layered on the insulator structure1290. A chemically sensitive mesh 1310 can be layered on the gate 1305.The present artificial receptors 1315 can be coupled to or embedded inone or more of the chemically sensitive mesh 1310, the gate 1305, andthe insulator structure 1290.

In an embodiment, the present artificial receptors can be coupled to acarbon paste that is electrically coupled to an electrode. In anembodiment, electrochemical sensors that incorporate the presentartificial receptors can be based on carbon paste screen-printedelectrodes. In an embodiment, these sensors incorporate the conductingpolymer polyaniline (PANI)/poly(vinylsulphonic acid) (PVSA). Suchsensors can be useful in detecting and quantifying a redox active testligand.

The present artificial receptors can also be configured withelectrochemical impedance spectroscopy techniques. In an embodiment, thepresence of a test ligand on a receptor can be detected through avariation in electrical properties at an electrode interface. Forexample, a shift in impedance, a change in capacitance, or a change inadmittance (or resistance) can be detected.

In an embodiment, the present artificial receptors can be configured inreagentless sensors. For example, the present artificial receptors canbe configured for use in reagentless amperometric immunosensors. In anembodiment, a present artificial receptor is fluorescent, and thefluorescent properties of the receptor change upon analyte binding. Areagentless assay kit is described in U.S. Pat. No. 6,660,532.

In an embodiment, optical and electrochemical sensors can be configuredto simultaneously gather data regarding a sample. A thin film fiberoptic sensor array and apparatus for concurrent viewing and chemicalsensing of a sample is described in U.S. Pat. No. 5,298,741.

The present artificial receptor can also be configured with amultiple-element microelectrode array sensor. Such array sensors can beconfigured, for example, to detect a variety of different gasses or testligands, or can monitor for a single test ligand at a variety oflocations.

FIG. 8 shows an embodiment of a sensing system 600 where an array ofelectrochemical sensors 620 and an array of fiber optic sensors in afiber cable 630 can be configured to detect signals from a multiplicityof present artificial receptors 610 that can be coupled to a substrate640. A processing system 650 receives data from the array ofelectrochemical sensors and from the optical sensors. An example of adata pattern 800 received from an array of fibers is shown in FIG. 10.Locations 810 where a test ligand has bound to a present artificialreceptor can be detected.

Sensor Applications

Sensors, sensor systems, and methods that employ the present artificialreceptors can be configured to detect a variety of test ligands ormeasure a variety of properties or behaviors.

Artificial Sense of Smell or Taste

In an embodiment, a sensor system that mimics a human or mammal sense ofsmell or taste can be provided using the present artificial receptors.Such systems are sometimes referred to as an artificial tongue orartificial nose. In an embodiment, for example, a sample can be testedfor bacteria, toxins, or poisons. In an embodiment, a system can beconfigured to detect, for example, a pathogenic microorganism, acancerous cell, a pollutant in water, an airborne pollutant, anexplosive-related vapor, protein, and/or a polynucleotide.

In an embodiment, the sample can be a food or beverage product. In anembodiment, an optical system can be configured to detect a change inoptical properties of a sample, such as a change in the intrinsicfluorescence of the sample. Data obtained through a fiber or fiberbundle can be deconvoluted or otherwise processed. Complex mixtures ofanalytes can be identified and quantified. For example, in anembodiment, a data pattern obtained from a sample can be interpreted toidentify and/or quantify the presence of a particular test ligand.

In an embodiment, the present artificial receptors can be configured inan artificial nose. For example, in an embodiment, the presentartificial receptors can be configured to detect explosives vapor, forexample to facilitate landmine detection. In an embodiment, anartificial nose includes a light source (or source of otherelectromagnetic radiation), an object to which the present artificialsensors can be coupled, and a CCD camera. In an embodiment, a vapordelivery system and a vapor removal system can also be provided. In anembodiment, vapors can be delivered to a sensing region of an artificialnose in pulses. To identify and quantify a test ligand in a sample,optical characteristics and behaviors such as change in fluorescenceand/or shift in wavelength can be detected.

In an embodiment of an artificial nose, pattern recognition and/orneural network analysis can be configured to discriminate betweenexplosives vapors and other organic vapors (e.g. background vapors.) Inan embodiment, an array of optical sensors can be used. In anembodiment, the present artificial receptors can be coupled to amicrobead that can be coupled to a microwell. In an embodiment,fluorescent signals can be processed and a signature can be mapped toallow identification, for example, of nitroamoratic compounds. In anembodiment, signals can be detected over a period of time and signaldata can be processed to identify and/or quantify the test ligand(s) ina sample. In an embodiment, an artificial nose based on the presentartificial receptors can be incorporated into a portable system that canbe configured for use in the field, for example to detect land mines.

Microfluidic Systems

The present artificial receptors can be configured for use withmicrofluidic systems. A biosensor incorporating a microfluidic system isdescribed in U.S. Pat. No. 6,716,620. Microfluidic systems are alsodescribed in U.S. Pat. Nos. 6,773,567; 6,756,019; 6,709,559; 6,670,153;6,670,133; 6,635,487; 6,632,655; 6,534,013; 6,498,353; 6,488,895; and6,465,257, and Published U.S. Patent Application Nos. 20040048360 and20040028567. In an embodiment, an optical sensor can be utilized todetect the presence of a test ligand in a portion of a microfluidicsystem. In an embodiment, the present artificial receptors can beemployed to extract a test ligand from a flow in a microfluidic system.Microfluidic systems can also be configured with indicator componentsusing the present artificial receptors. Indicator components formicrofluidic systems are described in Published U.S. Patent ApplicationNo. 20040141884.

Nanostructures

In an embodiment, the present artificial receptors can be configured foruse in nanostructures. In an embodiment, the present artificialreceptors can be configured for use in a colloidal assembly process. Inan embodiment, the receptors are coupled to a larger particle. Thecolloidal assembly process can be performed such that smaller particlesassemble around larger ones. In an embodiment, the larger particle canbe etched away to produce a hollow sphere.

In an embodiment, the present artificial receptors can be coupled tocantilevers, which can for example be micron-scale arms. When a testligand, a strand of DNA for example, bonds to the artificial receptors,a surface stress is induced which bends the cantilever. The bending ofthe cantilever arm can be detected, thereby allowing for detection ofthe presence of a test ligand bound to the present artificial receptor.In an embodiment, an array of cantilevers can be provided. In anembodiment, different test ligands can be detected (different genes forexample) by coupling different artificial receptors to cantilevers orgroups of canitilevers.

In an embodiment, the present artificial receptors can be configuredwith nanotubes. For example, the present artificial receptors can becoupled to a semiconducting carbon nanotube to facilitate detection ofgasses.

In an embodiment, the present artificial receptors can be configuredwith semiconducting nanotubes that are configured to change electricalresistance when exposed to a particular test ligand, such as a gas. Inan embodiment, the present artificial receptors are configured to blockthe binding of one or more test ligands with the a nanotube to allowdetection of other substances by the nanotube.

In an embodiment, the present artificial receptors can be coupled to thesurface of a semiconductor nanowire. For example the present artificialreceptors can be coupled to a nanowire field effect transistor. In anembodiment, the nanowire field effect transistor can be coupled to orintegrated into a silicon chip.

Sensor Coupled Communications Systems

Embodiments of the present artificial receptors can be configured with asensor that is operatively coupled to a communication system. Forexample, a sensor employing the present artificial receptors can becoupled to a communications network using wired or wireless technology.The communications can, for example, be the Internet. A processingsystem that is also coupled to the communications network can monitorone or more signals from one or more sensors. In an embodiment of aresponsive system, corrective action can be taken as necessary inresponse to signals received from a sensor.

Various types of sensors can be configured with a network. For example,electrochemical and fiber optic sensors can be configured with anetwork. In an embodiment, electrochemical, potentiometric, voltametric,and/or amperometric sensors that incorporate the present artificialreceptors can be coupled to a data network. Absorbance and/orfluorescence-based sensors that incorporate the present artificialreceptors can also be coupled to a data network. Other types of sensorsthat can include the present artificial receptors can also be coupled toa network. Systems can also be configured to gather and transmit datafrom a variety of sensors or sensor arrays.

In an embodiment, a sensor system can be coupled to a digital systemthat receives signals from the sensor system. In an embodiment, thedigital system can also be configured to make adjustments to anenvironment. For example, an actuator can be activated to make anadjustment. In an embodiment, an item for which particular propertiesare detected can be removed from a product flow. For example acontaminated food product can be removed from an assembly line. In anembodiment, a system can be configured to detect a particular testligand in a mail package, and the package can be isolated for processingor further analysis. In an embodiment, items such as aircraft luggagecan be monitored for explosive vapors and responsive action can beinitiated as necessary. Other embodiments are possible.

Methods Employing the Artificial Receptors

Working artificial receptors can be generated to be specific to a giventest ligand or specific to a particular part of a given test ligand.Heterogeneous and immobilized combinations of building block moleculesform the working artificial receptors. For example, combinations of 2,3, 4, or 5 distinct building block molecules immobilized in proximity toone another on a support provide molecular structures that serve ascandidate and working artificial receptors. The building blocks can benaïve to the test ligand. Once a plurality of candidate artificialreceptors are generated, they can be tested to determine which arespecific or useful for a given ligand.

The specific or working artificial receptor or receptor complex can thenbe used in a variety of different methods and systems. For example, thereceptors can be employed in methods and/or devices for binding ordetecting a test ligand. By way of further example, the receptors can beemployed in methods and/or devices for chemical synthesis. Methods andsystems for chemical synthesis can include methods and systems forregiospecific and stereospecific chemical synthesis. The receptors canalso be employed for developing compounds that disrupt or model bindinginteractions. Methods and systems for developing therapeutic agents caninclude methods and systems for pharmaceutical and vaccine development.

In an embodiment, methods and systems of the present invention can beemployed for detecting a plurality of ligands of interest. For example,an unknown biological sample can be characterized by the presence of acombination of specific ligands. Such a method can be useful in assaysfor detecting specific pathogens or disease states. By way of furtherexample, such an embodiment can be used for determining the geneticprofile of a subject. For example, cancerous tissue can be detected or agenetic disposition to cancer can be detected.

The present artificial receptors can be part of products used in:analyzing a genome and/or proteome (protein isolation andcharacterization); pharmaceutical development (such as identification ofsequence specific small molecule leads, characterization of protein toprotein interactions); detectors for a test ligand; drug of abusediagnostics or therapy (such as clinical or field analysis of cocaine orother drugs of abuse); hazardous waste analysis or remediation; chemicalexposure alert or intervention; disease diagnostics or therapy; cancerdiagnostics or therapy (such as clinical analysis of prostate specificantigen); biological agent alert or intervention; food chaincontamination analysis or remediation and clinical analysis of foodcontaminants; and the like.

Methods of Binding or Detecting Test Ligands

In an embodiment, the invention can include methods and/or devices forbinding or detecting a test ligand. For example, the present artificialreceptors can be used for a variety of assays that presently employ anantibody. The present artificial receptors can be specific for a givenligand, such as an antigen or an immunogen. Thus, the present artificialreceptors can be used in formats analogous to enzyme immunoassay,enzyme-linked immunoassay, immunodiffusion, immunoelectrophoresis, latexagglutination, and the like. Test ligands that can be detected in such amethod include a drug of abuse, a biological agent (such as a hazardousagent), a marker for a biological agent, a marker for a disease state,etc. Methods and systems for detection can include methods and systemsfor clinical chemistry, environmental analysis, and diagnostic assays ofall types.

For example, the artificial receptor can be contacted with a sampleincluding or suspected of including at least one test ligand. Thebuilding blocks making up the artificial receptors can be naïve to thetest ligand. Then, binding of one or more of the test ligands to theartificial receptors can be detected. Next, the binding results can beinterpreted to provide information about the sample. In an embodiment,the invention includes a method for detecting a test ligand in a sampleincluding contacting an artificial receptor specific to the test ligandwith a sample suspected of containing the test ligand. The method canalso include detecting or quantitating binding of the test ligand to theartificial receptor. For example, an artificial receptor that binds(e.g., tightly) the molecule, cell, or microbe under appropriateconditions can be employed in a format where binding itself issufficient to indicate presence of the molecule or organism. Such aformat can also include artificial receptors to be probed with positiveand control samples.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand,such as a molecule or cell. The method can include making an array ofcandidate artificial receptors. Working artificial receptors can beidentified by contacting the array with test ligand and identifyingwhich receptors bind the test ligand. The building blocks making up theartificial receptors can be naïve to the test ligand. Such a method canemploy a labeled test ligand. The method can include producing an arrayor device including the working artificial receptor or receptor complex.In an embodiment, the method can include employing the array or devicefor detecting or characterizing the test ligand in a sample, such as abiological, laboratory, or environmental sample.

FIG. 15 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors. This embodiment ofthe method can employ an array including a significant number of thepresent artificial receptors to produce an assay or system forcharacterizing or detecting a test ligand. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to a test ligand, e.g., a molecule orcell. The building blocks making up the artificial receptors can benaïve to the test ligand. The molecule or cell can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological sample, or characterizing ordetecting the molecule or cell.

As illustrated in FIG. 15, a test ligand can be identified by a methodemploying a single or a plurality of lead or working artificialreceptors. The plurality of lead or working artificial receptorssuitable for identifying a test ligand can be employed in an arrayformat test. A single lead or working artificial receptor can beconfigured on a support as a strip together with positive and/ornegative control receptors, which can also be configured as strips.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a single testligand or working artificial receptors for a plurality of test ligands.For example, a method can include contacting the artificial receptorswith a sample. A substrate including working artificial receptors for asingle test ligand can be employed in a method or system for detectingthat test ligand. Binding to the working artificial receptors indicatesthat the sample includes the test ligand. A substrate including workingartificial receptors for a plurality of test ligands can be employed ina method or system for detecting one, several, or all of the testligands. Binding to the working artificial receptors for a particulartest ligand or ligands indicates that the sample includes such testligand or ligands.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of thetest ligands. The method can include detecting the binding pattern ofthe sample and comparing it with binding patterns from known samples.FIG. 16 schematically illustrates certain binding patterns on an arrayof working artificial receptors. In an embodiment, all artificialreceptors for one test ligand can be arranged in a line across thesubstrate. Referring to FIG. 16, receptors that are specific for IL-2are in a line 12 on the array 10 of working artificial receptorcomplexes. Working artificial receptors that have bound a test ligand(e.g., IL-2) are indicated as shaded 24. Working artificial receptorsthat have not bound a test ligand are illustrated as open circles 26.

A method employing the illustrated array can include detecting bindingon line 12 of working artificial receptors through fluorescence oranother means described herein. In the illustrated embodiment, detectingbinding on line 12 of working artificial receptors indicates that thesample contains IL-2. Further, lack of signal from the other workingartificial receptors in array 10 indicates that the sample does notcontain IFN-gamma, IL-10, TGF-beta, IL-12, or TGF-alpha. Thus, a methodemploying such an array can determine whether a sample is a particulartype of biological sample or contains a particular type of molecule orcell.

When designed for use with a field assay kit, the device 30 can havespots arranged such that a positive result creates an easilyrecognizable pattern 36, such as a plus sign. The readily recognizablepattern can thus indicate that a particular test ligand is present inthe sample. Alternatively, the artificial receptors or spots for aparticular target 42 can be arranged randomly on third array 40. In thismanner, when the detection device or array is used, the results of thetest may not be immediately apparent to an observer but will be readilyread by a machine which can be programmed to correlate binding toreceptors or spots in different positions with the identity of aparticular biological sample, molecule, or cell.

In an embodiment, the invention includes a method for detecting orcharacterizing a biological sample, a molecule, or cell. This embodimentof the method can include selecting an artificial receptor that bindsthe biological sample, molecule, or cell from an array of artificialreceptors, contacting the artificial receptor with a test composition,and detecting binding of the artificial receptor to the testcomposition. In such an embodiment, binding indicates the presence ofthe biological sample, molecule, or cell in the test composition. In anembodiment, the invention includes a method for detecting orcharacterizing a biological sample, molecule, or cell. This embodimentof the method can include contacting an array of artificial receptorswith a test composition and detecting binding to the artificialreceptors. Binding indicates the presence of the biological sample,molecule, or cell in the test composition.

The present method can develop or employ a plurality of workingreceptors specific for a particular test ligand, e.g., biologicalsample, molecule, or cell. That is, the working receptors can bespecific for a particular test ligand, but different receptors caninteract with different distinct antigens (e.g., proteins orcarbohydrates), ligands, functional groups, or structural features ofthe test ligand. Such a method can provide a robust test for thepresence of a test ligand. For example, such a robust test can reducethe chances of a false-positive or false-negative result in comparisonwith an assay that relies upon a single unique receptor to detect agiven test ligand. Further, this embodiment of the method can develop oremploy working receptors that demonstrate higher binding affinity due tointeraction with multiple antigens or ligands on the same test ligand(e.g., multivalent binding).

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a polynucleotide, e.g., DNA or RNA. Themethod can include evaluating an array including a significant number ofcandidate artificial receptors for binding to the polynucleotide, e.g.,DNA or RNA. The building blocks making up the artificial receptors canbe naïve to the DNA or RNA. The polynucleotide, e.g., DNA or RNA, canexhibit characteristic binding to one or several of the candidateartificial receptors from that array. The one or several artificialreceptors can be selected as an artificial receptor (e.g., a workingartificial receptor or a working artificial receptor complex) that canbe employed in methods for characterizing a biological sample, orcharacterizing or detecting the polynucleotide, e.g., DNA or RNA.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a polypeptide or peptide. The method caninclude evaluating an array including a significant number of candidateartificial receptors for binding to the polypeptide or peptide. Thebuilding blocks making up the artificial receptors can be naïve to thepolypeptide or peptide. The polypeptide or peptide can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological sample, or characterizing ordetecting the polypeptide or peptide.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a oligo- or polysaccharide. The methodcan include evaluating an array including a significant number ofcandidate artificial receptors for binding to the oligo- orpolysaccharide. The building blocks making up the artificial receptorscan be naïve to the oligo- or polysaccharide. The oligo- orpolysaccharide can exhibit characteristic binding to one or several ofthe candidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sample,or characterizing or detecting the oligo- or polysaccharide.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a cell, e.g., a hepatocyte. The methodcan include evaluating an array including a significant number ofcandidate artificial receptors for binding to the cell, e.g., ahepatocyte. The building blocks making up the artificial receptors canbe naïve to the cell. The cell, e.g., a hepatocyte, can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological sample, or characterizing ordetecting the cell, e.g., a hepatocyte.

Methods of Binding or Detecting Drugs of Abuse

In an embodiment, the invention can include methods and/or devices forbinding or detecting a drug of abuse. Methods and systems for detectioncan include methods and systems for clinical chemistry, field analysis,and diagnostic assays of all types. For example, the artificial receptorcan be contacted with a sample including or suspected of including atleast one drug of abuse. Then, binding of one or more of the drugs ofabuse to the artificial receptors can be detected. Next, the bindingresults can be interpreted to provide information about the sample. Inan embodiment, the invention includes a method for detecting a drug ofabuse in a sample including contacting an artificial receptor specificto the drug of abuse with a sample suspected of containing the drug ofabuse. The method can also include detecting or quantitating binding ofthe drug of abuse to the artificial receptor.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand.This embodiment of the present method can be employed for detecting atest ligand such as a drug of abuse. The method can include making anarray of candidate artificial receptors. The building blocks making upthe artificial receptors can be naïve to the test ligand. Workingartificial receptors can be identified by contacting the array with adrug of abuse and identifying which receptors bind the drug of abuse.The method can include producing an array or device including theworking artificial receptor or receptor complex. In an embodiment, themethod can include employing the array or device for detecting orcharacterizing the drug of abuse in a sample, such as a biological,laboratory, or evidence sample.

FIG. 15 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors. This embodiment ofthe method can employ an array including a significant number of thepresent artificial receptors to produce an assay or system forcharacterizing or detecting a drug of abuse. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to a drug of abuse. The building blocksmaking up the artificial receptors can be naïve to the drug of abuse.The drug of abuse can exhibit characteristic binding to one or severalof the candidate artificial receptors from that array. The one orseveral artificial receptors can be selected as an artificial receptor(e.g., a working artificial receptor or a working artificial receptorcomplex) that can be employed in methods for characterizing a biologicalor field sample, or characterizing or detecting the drug of abuse.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a single drugof abuse or working artificial receptors for a plurality of drugs ofabuse. For example, a method can include contacting the artificialreceptors with a sample. A substrate including working artificialreceptors for a single drug of abuse can be employed in a method orsystem for detecting that drug of abuse. Binding to the workingartificial receptors indicates that the sample includes the drug ofabuse. A substrate including working artificial receptors for aplurality of drugs of abuse can be employed in a method or system fordetecting one, several, or all of the drugs of abuse. Binding to theworking artificial receptors for a particular drug of abuse or drugs ofabuse indicates that the sample includes such a drug of abuse or drugsof abuse.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of thedrugs of abuse. The method can include detecting the binding pattern ofthe sample and comparing it with binding patterns from known samples.FIG. 16 schematically illustrates binding patterns on an array ofworking artificial receptors. Such patterns and schemes can be employedfor identifying a variety of test ligands including drugs of abuse.

The present method can develop or employ a plurality of workingreceptors specific for a particular drug of abuse or feature on the drugof abuse. That is, the working receptors can be specific for aparticular drug of abuse, but different receptors can interact withdifferent distinct ligands, functional groups, or structural features ofthe drug of abuse. Such a method can provide a robust test for thepresence of a drug of abuse. For example, such a robust test can reducethe chances of a false-positive or false-negative result in comparisonwith an assay that relies upon a single unique receptor to detect agiven drug of abuse. Further, this embodiment of the method can developor employ working receptors that demonstrate higher binding affinity dueto interaction with multiple ligands or features on the same drug ofabuse (e.g., multivalent binding).

Suitable drugs of abuse include cannabinoids (e.g., hashish andmarijuana), depressants (e.g., barbiturates, benzodiazepines,gamma-hydroxy butyrate, methaqualone), dissociative anesthetics (e.g.,ketamine, PCP, and PCP analogs), hallucinogens (e.g., LSD, mescaline,psilocybin), opiates or opioids (e.g., codeine, fentanyl, fentanylanalogs, heroin, morphine, opium, oxycodone HCL, hydrocodonebitartrate), stimulants (e.g., amphetamine, cocaine,methylenedioxy-methamphetamine, methamphetamine, methylphenidate,nicotine), inhalants (e.g., solvents), and the like.

Suitable drugs of abuse include performance enhancing agents, such asstimulants and beta-blockers, anabolic agents, oxygen carrier enhancers,masking agents, and inhalants. Suitable stimulants include caffeine andamphetamines. Suitable beta-blockers include salbutamol (used in asthmainhalers) and the like. Suitable anabolic agents include steroids (e.g.,anabolic steroids), steroid analogs, and growth hormone. Suitable oxygencarrier enhancers include erythropoietin and the like.

Methods of Binding or Detecting Isomers

In an embodiment, the invention can include methods and/or devices forbinding or detecting an isomer or isomers of a compound. Methods andsystems for detection can include methods and systems for clinicalchemistry, environmental analysis, and diagnostic assays of all types.For example, the artificial receptor can be contacted with a sampleincluding or suspected of including at least one isomer of a compound.Then, binding of one or more of the isomers of a compound to theartificial receptors can be detected. Next, the binding results can beinterpreted to provide information about the isomers. In an embodiment,the invention includes a method for detecting an isomer of a compound ina sample including contacting an artificial receptor specific to theisomer with a sample suspected of containing the isomer. The method canalso include detecting or quantitating binding of the isomer to theartificial receptor.

The present method can be applied to isomers such as stereoisomers(e.g., geometric isomers or optical isomers), optical isomers (e.g.,enantiomers and diastereomers), geometric isomers (e.g., cis- andtrans-isomers). The present method can be employed to develop working orlead artificial receptors or working artificial complexes that can bindto one or more isomers of a compound (e.g., enantioselective receptorenvironments). For example, the artificial receptor or complex can bindto one stereoisomer of a compound but bind only weakly or not at allanother stereoisomer of the compound. For example, the artificialreceptor or complex can bind one geometric isomer of a compound but bindonly weakly or not at all another geometric isomer. For example, theartificial receptor or complex can bind one optical isomer of a compoundbut bind only weakly or not at all another optical isomer. For example,the artificial receptor or complex can bind one enantiomer of a compoundbut bind only weakly or not at all another enantiomer. For example, theartificial receptor or complex can bind one diastereomer of a compoundbut bind only weakly or not at all another diastereomer.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand.This embodiment of the present method can be employed for detecting atest ligand such as an isomer of a compound. The method can includemaking an array of candidate artificial receptors. The building blocksmaking up the artificial receptors can be naïve to the test ligand.Working artificial receptors can be identified by contacting the arraywith an isomer and identifying which receptors bind the isomer. Themethod can include producing an array or device including the workingartificial receptor or receptor complex. In an embodiment, the methodcan include employing the array or device for detecting orcharacterizing the isomer in a sample, such as a biological, laboratory,or clinical sample.

FIG. 15 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors. This embodiment ofthe method can employ an array including a significant number of thepresent artificial receptors to produce an assay or system forcharacterizing or detecting an isomer. The method can include evaluatingan array including a significant number of candidate artificialreceptors for binding to an isomer. The building blocks making up theartificial receptors can be naïve to the isomer. The isomer can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological, clinical, or laboratorysample, or characterizing or detecting the isomer.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a singleisomer or working artificial receptors for a plurality of isomers. Forexample, a method can include contacting the artificial receptors with asample. A substrate including working artificial receptors for a singleisomer can be employed in a method or system for detecting that isomer.Binding to the working artificial receptors indicates that the sampleincludes the isomer. A substrate including working artificial receptorsfor a plurality of isomers can be employed in a method or system fordetecting one, several, or all of the isomers. Binding to the workingartificial receptors for a particular isomer or isomers indicates thatthe sample includes such an isomer or isomers.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of theisomers. The method can include detecting the binding pattern of thesample and comparing it with binding patterns from known samples. FIG.16 schematically illustrates binding patterns on an array of workingartificial receptors. Such patterns and schemes can be employed foridentifying a variety of test ligands including isomers.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a stereoisomer. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the stereoisomer. The buildingblocks making up the artificial receptors can be naïve to thestereoisomer. The stereoisomer can exhibit characteristic binding to oneor several of the candidate artificial receptors from that array. Theone or several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing alab or clinical sample or characterizing or detecting the stereoisomer.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a geometric isomer (e.g., cis- andtrans-isomers). The method can include evaluating an array including asignificant number of candidate artificial receptors for binding to thegeometric isomer (e.g., cis- and trans-isomers). The building blocksmaking up the artificial receptors can be naïve to the geometric isomer.The geometric isomer can exhibit characteristic binding to one orseveral of the candidate artificial receptors from that array. The oneor several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing alab or clinical sample or characterizing or detecting the geometricisomer (e.g., cis- and trans-isomers).

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting an optical isomer. The method caninclude evaluating an array including a significant number of candidateartificial receptors for binding to the optical isomer. The buildingblocks making up the artificial receptors can be naïve to the opticalisomer. The optical isomer can exhibit characteristic binding to one orseveral of the candidate artificial receptors from that array. The oneor several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing alab or clinical sample or characterizing or detecting the opticalisomer.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting an enantiomer. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the enantiomer. The enantiomer canexhibit characteristic binding to one or several of the candidateartificial receptors from that array. The one or several artificialreceptors can be selected as an artificial receptor (e.g., a workingartificial receptor or a working artificial receptor complex) that canbe employed in methods for characterizing a lab or clinical sample orcharacterizing or detecting the enantiomer.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a diastereomer. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the diastereomer. The buildingblocks making up the artificial receptors can be naïve to thediastereomer. The diastereomer can exhibit characteristic binding to oneor several of the candidate artificial receptors from that array. Theone or several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing alab or clinical sample or characterizing or detecting the diastereomer.

Methods for Binding or Detecting Peptides

In an embodiment, the invention can include methods and/or devices forbinding or detecting a peptide. Methods and systems for detection caninclude methods and systems for clinical chemistry, environmentalanalysis, and diagnostic assays of all types. For example, theartificial receptor can be contacted with a sample including orsuspected of including at least one peptide. Then, binding of one ormore of the peptides to the artificial receptors can be detected. Next,the binding results can be interpreted to provide information about thesample. In an embodiment, the invention includes a method for detectinga peptide in a sample including contacting an artificial receptorspecific to the peptide with a sample suspected of containing thepeptide. The method can also include detecting or quantitating bindingof the peptide to the artificial receptor.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand.This embodiment of the present method can be employed for detecting atest ligand such as a peptide. The method can include making an array ofcandidate artificial receptors. The building blocks making up theartificial receptors can be naïve to the test ligand. Working artificialreceptors can be identified by contacting the array with a peptide andidentifying which receptors bind the peptide. The method can includeproducing an array or device including the working artificial receptoror receptor complex. In an embodiment, the method can include employingthe array or device for detecting or characterizing the peptide in asample, such as a biological, laboratory, or clinical sample.

FIG. 15 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors. This embodiment ofthe method can employ an array including a significant number of thepresent artificial receptors to produce an assay or system forcharacterizing or detecting a peptide. The method can include evaluatingan array including a significant number of candidate artificialreceptors for binding to a peptide. The building blocks making up theartificial receptors can be naïve to the peptide. The peptide canexhibit characteristic binding to one or several of the candidateartificial receptors from that array. The one or several artificialreceptors can be selected as an artificial receptor (e.g., a workingartificial receptor or a working artificial receptor complex) that canbe employed in methods for characterizing a biological or environmentalsample, or characterizing or detecting the peptide.

FIG. 17 schematically illustrates an embodiment of a method fordeveloping a method and system for detecting a test ligand, such as apeptide or mixture of peptides. This embodiment of the present methodincludes evaluating a plurality (e.g. array) of candidate artificialreceptors for binding to each of a plurality of peptides. The buildingblocks making up the artificial receptors can be naïve to one or more ofthe peptides. The plurality of peptides can include the peptides foundin a cell or organism. The method can include detecting binding ofindividual peptides to a subset of the plurality or array of candidateartificial receptors. The method can include detecting binding of thepeptides found in a cell or organism to a subset of or all of theplurality or array of candidate artificial receptors. This can beenvisioned as developing a working artificial receptor or artificialreceptor complex for each peptide or mixture of peptides.

Thus, each peptide or mixture of peptides can provide a pattern of boundreceptors in the plurality or array. The pattern of bound receptors canbe characteristic of the peptide or mixture of peptides or a sampleincluding the peptide or mixture of peptides. The method can includestoring a representation of the binding pattern as an image or a datastructure. The representation of the binding pattern can be evaluatedeither by an operator or data processing system. The method can includesuch evaluating. A binding pattern from an unknown sample that matchesthe binding pattern for a particular peptide then characterizes theunknown sample as containing that peptide. A binding pattern from anunknown sample that matches the binding pattern for a particular mixtureof peptides then characterizes the unknown sample as including or beingthat mixture of peptides or as including or being the organism or cellcontaining that mixture of peptides. A plurality of binding patterns canbe stored as a database.

An embodiment of the illustrated method can include creating an array ofartificial receptors. This embodiment can also include compiling adatabase of the binding patterns of a specific peptide or mixture ofpeptides, for example, by probing the array with a plurality ofindividual peptides or the peptides found in a cell or organism.Contacting the array with an unidentified peptide or mixture of peptidescan create a test binding pattern. The method can then compare the testbinding pattern with the binding patterns of known peptides or mixturesof peptides in the database in order to characterize or classify theunidentified peptide, mixture of peptides, or cell or organism. In anembodiment, the database and the array of receptors has already beenconstructed and the method involves probing the array with an unknownpeptide or mixture of peptides to create a test binding pattern and thencomparing this binding pattern with the binding patterns in the databasein order to characterize or classify the unidentified peptide, mixtureof peptides, or cell or organism.

An array constructed for distinguishing mixtures of peptides can becontacted with samples from an organism, cell, or tissue of interest.Peptides that bind to the array can characterize or detect the organism,cell or tissue; can indicate a disorder caused by the organism oraffecting the cell or tissue; can indicate successful therapy of adisorder caused by the organism or affecting the cell or tissue;characterize disease processes; identify therapeutic leads orstrategies; or the like.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a singlepeptide or working artificial receptors for a plurality of peptides. Forexample, a method can include contacting the artificial receptors with asample. A substrate including working artificial receptors for a singlepeptide can be employed in a method or system for detecting thatpeptide. Binding to the working artificial receptors indicates that thesample includes the peptide. A substrate including working artificialreceptors for a plurality of peptides can be employed in a method orsystem for detecting one, several, or all of the peptides. Binding tothe working artificial receptors for a particular peptide or peptidesindicates that the sample includes such a peptide or peptides.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of thepeptides. The method can include detecting the binding pattern of thesample and comparing it with binding patterns from known samples. FIG.16 schematically illustrates binding patterns on an array of workingartificial receptors. Such patterns and schemes can be employed foridentifying a variety of test ligands including peptides.

The present method can develop or employ a plurality of workingreceptors specific for a particular peptide or feature on the peptide.That is, the working receptors can be specific for a particular peptide,but different receptors can interact with different distinct ligands,functional groups, or structural features of the peptide. Such a methodcan provide a robust test for the presence of a peptide. For example,such a robust test can reduce the chances of a false-positive orfalse-negative result in comparison with an assay that relies upon asingle unique receptor to detect a given peptide. Further, thisembodiment of the method can develop or employ working receptors thatdemonstrate higher binding affinity due to interaction with multipleligands or features on the same peptide (e.g., multivalent binding).

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a peptide. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the peptide. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Thepeptide can exhibit characteristic binding to one or several of thecandidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sampleor characterizing or detecting the peptide.

The present method can include selecting artificial receptors that binda particular peptide and/or the building blocks making up thesereceptors (e.g., bound to a scaffold molecule) as leads forpharmaceutical development or as active agents for modulating anactivity of that peptide. The artificial receptor or building blocksmaking up that artificial receptor can be selected to bind to a portionof a peptide required for its interaction with an other macromolecule(e.g. carbohydrate, protein, or polynucleotide), thus disrupting thisinteraction.

Methods for Binding or Detecting Protein or Proteome

In an embodiment, the invention can include methods and/or devices forbinding or detecting a protein, one or more of a plurality of proteins,or a proteome. Methods and systems for detection can include methods andsystems for clinical chemistry, environmental analysis, diagnosticassays, and for proteome analysis. For example, the artificial receptorcan be contacted with a sample including at least one protein or oneproteome. The building blocks making up the artificial receptors can benaïve to the test ligand. Then, binding of one or more proteins to theartificial receptors can be detected. Next, the binding results can beinterpreted to provide information about the sample, e.g., the proteome.In an embodiment, the invention includes a method for detecting aprotein in a sample including contacting an artificial receptor specificto the protein with a sample suspected of containing the protein. Themethod can also include detecting or quantitating binding of the proteinto the artificial receptor.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand.This embodiment of the present method can be employed for detecting atest ligand such as one or more proteins. The method can include makingan array of candidate artificial receptors. The building blocks makingup the artificial receptors can be naïve to the test ligand. Workingartificial receptors can be identified by contacting the array with aprotein and identifying which receptors bind the protein. The method caninclude producing an array or device including the working artificialreceptor or receptor complex. In an embodiment, the method can includeemploying the array or device for detecting or characterizing theprotein in a sample, such as a biological, laboratory, or environmentalsample.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a singleprotein or working artificial receptors for a plurality of proteins. Forexample, a method can include contacting the artificial receptors with asample. A substrate including working artificial receptors for a singleprotein can be employed in a method or system for detecting thatprotein. Binding to the working artificial receptors indicates that thesample includes the protein. A substrate including working artificialreceptors for a plurality of proteins can be employed in a method orsystem for detecting one, several, or all of the proteins. Binding tothe working artificial receptors for a particular protein or proteinindicates that the sample includes such a protein or protein.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of theproteins. The method can include detecting the binding pattern of thesample and comparing it with binding patterns from known samples. FIG.16 schematically illustrates binding patterns on an array of workingartificial receptors. Such patterns and schemes can be employed foridentifying a variety of test ligands including proteins.

FIG. 17 schematically illustrates an embodiment of a method fordeveloping a method and system for detecting a test ligand, such as aprotein or proteome. This embodiment of the present method includesevaluating a plurality (e.g. array) of candidate artificial receptorsfor binding to each of a plurality of test ligands. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Theplurality of test ligands can include a plurality of proteins. Theplurality of test ligands can include the proteins making up theproteome of a cell or organism. The method can include detecting bindingof individual proteins to a subset of the plurality or array ofcandidate artificial receptors. The method can include detecting bindingof proteins making up the proteome to a subset of or all of theplurality or array of candidate artificial receptors. This can beenvisioned as developing a working artificial receptor or artificialreceptor complex for each protein or for the proteome.

Thus, each protein or proteome can provide a pattern of bound receptorsin the plurality or array. The pattern of bound receptors can becharacteristic of the protein or proteome or a sample including theprotein or proteome. The method can include storing a representation ofthe binding pattern as an image or a data structure. The representationof the binding pattern can be evaluated either by an operator or dataprocessing system. The method can include such evaluating. A bindingpattern from an unknown sample that matches the binding pattern for aparticular protein then characterizes the unknown sample as containingthat protein. A binding pattern from an unknown sample that matches thebinding pattern for a particular proteome then characterizes the unknownsample as including or being that proteome or as including or being theorganism or cell having that proteome. Similarly, a binding pattern froman unknown sample can be evaluated against the patterns of a pluralityof particular proteins or proteomes and the sample can be characterizedas containing one or more of the proteins or proteomes. A plurality ofbinding patterns can be stored as a database.

An embodiment of the illustrated method can include creating an array ofartificial receptors. This embodiment can also include compiling adatabase of the binding patterns of specific proteins or proteomes, forexample, by probing the array with a plurality of individual proteins orproteomes. Contacting the array with unidentified proteins or proteomescan create a test binding pattern. The method can then compare the testbinding pattern with the binding patterns of known proteins or proteomesin the database in order to characterize or classify the unidentifiedprotein, proteome, or cell or organism. In an embodiment, the databaseand the array of receptors has already been constructed and the methodinvolves probing the array with an unknown protein or proteome to createa test binding pattern and then comparing this binding pattern with thebinding patterns in the database in order to characterize or classifythe unidentified protein, proteome, or cell or organism.

A proteome array can be contacted with samples from an organism, cell,or tissue of interest. Proteins that bind to the proteome array cancharacterize or detect the organism, cell or tissue; can indicate adisorder caused by the organism or affecting the cell or tissue; canindicate successful therapy of a disorder caused by the organism oraffecting the cell or tissue; characterize disease processes; identifytherapeutic leads or strategies; or the like.

The present method can develop or employ a plurality of workingreceptors specific for a particular protein or feature on the protein.That is, the working receptors can be specific for a particular protein,but different receptors can interact with different distinct ligands,functional groups, or structural features of the protein. Such a methodcan provide a robust test for the presence of a protein. For example,such a robust test can reduce the chances of a false-positive orfalse-negative result in comparison with an assay that relies upon asingle unique receptor to detect a given protein. Further, thisembodiment of the method can develop or employ working receptors thatdemonstrate higher binding affinity due to interaction with multipleligands or features on the same protein (e.g., multivalent binding).

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a protein. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the protein. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Theprotein can exhibit characteristic binding to one or several of thecandidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sampleor characterizing or detecting the protein.

The present method can include selecting artificial receptors that binda particular protein and/or the building blocks making up thesereceptors (e.g., bound to a scaffold molecule) as leads forpharmaceutical development or as active agents for modulating anactivity of that protein. The artificial receptor or building blocksmaking up that artificial receptor can be selected to bind to or disruptthe activity of the active site of an enzyme or the ligand binding siteof a receptor. The artificial receptor or building blocks making up thatartificial receptor can be selected to bind to a portion of a proteinrequired for its interaction with an other macromolecule (e.g.carbohydrate, protein, or polynucleotide), thus disrupting thisinteraction. The artificial receptor or building blocks making up thatartificial receptor can be selected to bind to the binding site of areceptor and act as an agonist of that receptor.

The present method can include selecting working artificial receptorsthat bind a preselected protein for use in a system for proteomeanalysis. The working artificial receptors for the preselected proteincan be provided on a substrate and the protein bound to the receptors.In an embodiment, selecting and binding employ a plurality of differentworking artificial receptors for the preselected protein. The pluralityof artificial receptors may bind to different features on thepreselected protein and leave free different features on the preselectedprotein. This embodiment of the method includes contacting the workingreceptors with bound preselected protein to at least one candidatebinding partner for the preselected protein. The method can includedetecting binding or absence of binding of the candidate binding partnerto the preselected protein. A candidate binding partner that binds tothe preselected protein can be considered a lead binding partner.

In an embodiment, the method includes contacting the working receptorswith bound preselected protein with a proteome of a cell or organismserving as the source of candidate binding partners. The method can thenrecover from the proteome one or more lead binding partners. This canthen characterize the proteome as containing or not a binding partnerfor the preselected protein.

In an embodiment, the present artificial receptors can be employed instudies of proteomics. In such an embodiment, an array of candidate orworking artificial receptors can be contacted with a mixture ofpeptides, polypeptides, and/or proteins. Each mixture can produce acharacteristic fingerprint of binding to the array. In addition,identification of a specific receptor environment for a target peptide,polypeptide, and/or protein can be utilized for isolation and analysisof the target. That is, in yet another embodiment, a particular receptorsurface can be employed for affinity purification methods, e.g. affinitychromatography.

In an embodiment, the present candidate artificial receptors can beemployed to find receptor surfaces that bind proteins in a preferredconfiguration or orientation. Many proteins (e.g. antibodies, enzymes,receptors) are stable and/or active in specific environments. Definedreceptor surfaces can be used to produce binding environments thatselectively retain or orient the protein for maximum stability and/oractivity. In an embodiment, the present artificial receptors can beemployed to form bioactive surfaces. For example, receptor surfaces canbe used to specifically bind the active conformation of an antibody orenzyme.

In an embodiment, the present method can include labeling a proteinwhile it remains bound to an artificial receptor. The resulting proteinwill be labeled on its portions accessible to the labeling reagent butnot on those portions bound to the artificial receptor. The method caninclude releasing the labeled protein from the artificial receptor.Determining the distribution of labels on the protein indicates whichportion of the protein was bound to the receptor.

In certain embodiments, the present artificial receptors can be employedto distinguish between two conformations of a single protein. Certainproteins exist in two or more stable conformations. In an embodiment,the present working artificial receptor or complex can bind a firstconformation of a protein. In an embodiment, the present workingartificial receptor or complex can bind a second conformation of aprotein. In an embodiment, the present working artificial receptor orcomplex can bind a first conformation of a protein, but not a secondconformation of the same protein. In an embodiment, the present workingartificial receptor or complex can bind a second conformation of aprotein, but not a first conformation of the same protein.

For example, in an embodiment, the present working artificial receptoror complex can bind a first or non-infectious conformation of a prion,but not its second or infectious conformation. For example, in anembodiment, the present working artificial receptor or complex can bindthe second or infectious conformation of a prion, but not its first ornon-infectious conformation. For example, in an embodiment, the presentworking artificial receptor or complex can bind a first ornon-plaque-forming conformation of β-amyloid, but not its second orplaque-forming conformation. For example, in an embodiment, the presentworking artificial receptor or complex can bind a second orplaque-forming conformation of β-amyloid, but not its second ornon-plaque-forming conformation.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a desired conformation of a protein. Themethod can include evaluating an array including a significant number ofcandidate artificial receptors for binding to the desired conformationof the protein. The building blocks making up the artificial receptorscan be naïve to the protein or its desired conformation. The desiredconformation of the protein can exhibit characteristic binding to one orseveral of the candidate artificial receptors from that array. The oneor several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing abiological sample or characterizing or detecting the desiredconformation of the protein.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a first or non-infectious conformationof a prion. The method can include evaluating an array including asignificant number of candidate artificial receptors for binding to thefirst or non-infectious conformation of a prion. The building blocksmaking up the artificial receptors can be naïve to the prion. The firstor non-infectious conformation of a prion can exhibit characteristicbinding to one or several of the candidate artificial receptors fromthat array. The one or several artificial receptors can be selected asan artificial receptor (e.g., a working artificial receptor or a workingartificial receptor complex) that can be employed in methods forcharacterizing a biological sample or characterizing or detecting thefirst or non-infectious conformation of a prion.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a second or infectious conformation of aprion. The method can include evaluating an array including asignificant number of candidate artificial receptors for binding to thesecond or infectious conformation of a prion. The building blocks makingup the artificial receptors can be naïve to the test ligand. The secondor infectious conformation of a prion can exhibit characteristic bindingto one or several of the candidate artificial receptors from that array.The one or several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing abiological sample or characterizing or detecting the second orinfectious conformation of a prion.

In an embodiment, the present method includes developing receptors or areceptor system that can distinguish between the first or non-infectiousconformation of a prion and the second or infectious conformation of theprion. Such a method can include selecting a working artificial receptoror complex can that bind the first or non-infectious conformation of aprion, but not the second or infectious conformation of the prion. Thisembodiment can include selecting a working artificial receptor orcomplex can that bind the second or infectious conformation of a prion,but not the first or non-infectious conformation of the prion. Employedtogether, these two sets of working artificial receptors or systems cancharacterize a biological sample as containing one or both forms of theprion.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a first or non-plaque-formingconformation of a β-amyloid. The method can include evaluating an arrayincluding a significant number of candidate artificial receptors forbinding to the first or non-plaque-forming conformation of theβ-amyloid. The building blocks making up the artificial receptors can benaïve to the β-amyloid. The first or non-plaque-forming conformation ofthe β-amyloid can exhibit characteristic binding to one or several ofthe candidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sampleor characterizing or detecting the first or non-plaque-formingconformation of the β-amyloid.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a second or plaque-forming conformationof a β-amyloid. The method can include evaluating an array including asignificant number of candidate artificial receptors for binding to thesecond or plaque-forming conformation of a β-amyloid. The buildingblocks making up the artificial receptors can be naïve to the β-amyloid.The second or plaque-forming conformation of the β-amyloid can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological sample or characterizing ordetecting the second or plaque-forming conformation of the β-amyloid.

In an embodiment, the present method includes developing receptors or areceptor system that can distinguish between the first ornon-plaque-forming conformation of β-amyloid and the second orplaque-forming conformation of the β-amyloid. Such a method can includeselecting a working artificial receptor or complex can that bind thefirst or non-plaque-forming conformation of β-amyloid, but not thesecond or plaque-forming conformation of the β-amyloid. This embodimentcan include selecting a working artificial receptor or complex can thatbind the second or plaque-forming conformation of the β-amyloid, but notthe first or non-plaque-forming conformation of β-amyloid. Employedtogether, these two sets of working artificial receptors or systems cancharacterize a biological sample as containing one or both forms of theβ-amyloid.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting cholera toxin. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the cholera toxin. The buildingblocks making up the artificial receptors can be naïve to the testligand. The cholera toxin can exhibit characteristic binding to one orseveral of the candidate artificial receptors from that array. The oneor several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing abiological sample, or characterizing or detecting cholera toxin.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting at least one protein of a cancer cell.The method can include evaluating an array including a significantnumber of candidate artificial receptors for binding to the cancer cellprotein. The building blocks making up the artificial receptors can benaïve to the test ligand. The cancer cell protein can exhibitcharacteristic binding to one or several of the candidate artificialreceptors from that array. The one or several artificial receptors canbe selected as an artificial receptor (e.g., a working artificialreceptor or a working artificial receptor complex) that can be employedin methods for characterizing a biological sample or characterizing ordetecting the cancer cell protein.

In an embodiment, the present method can include contacting a workingartificial receptor or array with a sample from cells or tissuessuspected of being cancerous or including a tumor. The sample can beserum. Binding of at least one protein to the working artificialreceptor or array can indicate or characterize the presence of theparticular cancer or tumor, such as by characterizing the pattern ofproteins present.

Cancers that can be detected or characterized by such a method include,for example, bladder cancer, breast cancer, colon cancer, kidney cancer,liver cancer, lung cancer, including small cell lung cancer, esophagealcancer, gall-bladder cancer, ovarian cancer, pancreatic cancer, stomachcancer, cervical cancer, thyroid cancer, prostate cancer, and skincancer, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin'slymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett'slymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias, myelodysplastic syndrome andpromyelocytic leukemia; tumors of mesenchymal origin, includingfibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheralnervous system, including astrocytoma, neuroblastoma, glioma andschwannomas; other tumors, including melanoma, seminoma,teratocarcinoma, osteosarcoma, xeroderoma pigmentosum, keratoctanthoma,thyroid follicular cancer, Kaposi's sarcoma, and the like.

Methods of Binding or Detecting Microbes

In an embodiment, the invention can include methods and/or devices forbinding or detecting a microbe, e.g., cell or virus. Methods and systemsfor detection can include methods and systems for clinical chemistry,environmental analysis, and diagnostic assays of all types. For example,the artificial receptor can be contacted with a sample including orsuspected of including at least one microbe, e.g., cell or virus. Thebuilding blocks making up the artificial receptors can be naïve to thetest ligand. Then, binding of one or more of the microbes to theartificial receptors can be detected. Next, the binding results can beinterpreted to provide information about the sample. In an embodiment,the invention includes a method for detecting a microbe, e.g., cell orvirus, in a sample including contacting an artificial receptor specificto the microbe, e.g., cell or virus, with a sample suspected ofcontaining the microbe, e.g., cell or virus. The method can also includedetecting or quantitating binding of the microbe, e.g., cell or virus,to the artificial receptor.

FIG. 14 schematically illustrates an embodiment of a method forevaluating candidate artificial receptors for binding to a test ligand.This embodiment of the present method can be employed for detecting atest ligand such as a microbe, e.g., cell or virus. The method caninclude making an array of candidate artificial receptors. The buildingblocks making up the artificial receptors can be naïve to the testligand. Working artificial receptors can be identified by contacting thearray with a microbe, e.g., cell or virus, and identifying whichreceptors bind the microbe. The method can include producing an array ordevice including the working artificial receptor or receptor complex. Inan embodiment, the method can include employing the array or device fordetecting or characterizing the microbe, e.g., cell or virus, in asample, such as a biological, laboratory, or environmental sample.

FIG. 15 schematically illustrates an embodiment of the present methodemploying an array of candidate artificial receptors. This embodiment ofthe method can employ an array including a significant number of thepresent artificial receptors to produce an assay or system forcharacterizing or detecting a microbe, e.g., cell or virus. The methodcan include evaluating an array including a significant number ofcandidate artificial receptors for binding to a microbe, e.g., cell orvirus. The building blocks making up the artificial receptors can benaïve to the test ligand. The microbe can exhibit characteristic bindingto one or several of the candidate artificial receptors from that array.The one or several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing abiological sample, or characterizing or detecting the microbe, e.g.,cell or virus.

FIG. 17 schematically illustrates an embodiment of a method fordeveloping a method and system for detecting a test ligand, such as adisease causing organism. This embodiment of the present method includesevaluating a plurality (e.g. array) of candidate artificial receptorsfor binding to each of a plurality of test ligands, such as diseasecausing organisms. The building blocks making up the artificialreceptors can be naïve to the test ligands. The method can includedetecting binding of each test ligand (e.g., disease causing organism)to a subset of the plurality or array of candidate artificial receptors.This can be envisioned as developing a working artificial receptor orartificial receptor complex for each of the plurality of test ligands.

Thus, each test ligand (e.g., disease causing organism) can provide apattern of bound receptors in the plurality or array. The pattern ofbound receptors can be characteristic of the test ligand or a sampleincluding the test ligand. The method can include storing arepresentation of the binding pattern as an image or a data structure.The representation of the binding pattern can be evaluated either by anoperator or data processing system. The method can include suchevaluating. A binding pattern from an unknown sample that matches thebinding pattern for a particular test ligand (e.g., disease causingorganism) then characterizes the unknown sample as containing that testligand. Similarly, a binding pattern from an unknown sample can beevaluated against the patterns of a plurality of particular test ligandsand the sample can be characterized as containing one or more of thetest ligands. A plurality of binding patterns can be stored as adatabase.

An embodiment of the illustrated method can include creating an array ofartificial receptors. This embodiment can also include compiling adatabase of the binding patterns of specific disease causing organisms,for example, by probing the array with a plurality of individualorganisms. Contacting the array with an unidentified organism can createa test binding pattern. The method can then compare the test bindingpattern with the binding patterns of known organisms in the database inorder to characterize or classify the unidentified organism. In anembodiment, the database and the array of receptors has already beenconstructed and the method involves probing the array with an unknownorganism to create a test binding pattern and then comparing thisbinding pattern with the binding patterns in the database in order tocharacterize or classify the unidentified organism.

In an embodiment, the method can include producing or employing theselected working artificial receptor or receptor complex on a substrate.The substrate can include working artificial receptors for a singlemicrobe, e.g., cell or virus, or working artificial receptors for aplurality of microbes, e.g., cells or viruses. For example, a method caninclude contacting the artificial receptors with a sample. A substrateincluding working artificial receptors for a single microbe, e.g., cellor virus, can be employed in a method or system for detecting thatmicrobe. Binding to the working artificial receptors indicates that thesample includes the microbe. A substrate including working artificialreceptors for a plurality of microbes, e.g., cells or viruses can beemployed in a method or system for detecting one, several, or all of themicrobes. Binding to the working artificial receptors for a particularmicrobe or microbes indicates that the sample includes such a microbe ormicrobes.

The working artificial receptors or receptor complexes can be configuredto provide a pattern indicative of the presence of one or more of themicrobes, e.g., cells or viruses. The method can include detecting thebinding pattern of the sample and comparing it with binding patternsfrom known samples. FIG. 16 schematically illustrates binding patternson an array of working artificial receptors. Such patterns and schemescan be employed for identifying a variety of test ligands includingmicrobes.

The present method can develop or employ a plurality of workingreceptors specific for a particular microbe or feature on the microbe.That is, the working receptors can be specific for a particular microbe,but different receptors can interact with different distinct antigens(e.g., proteins or carbohydrates), ligands, or features of the microbe.Such a method can provide a robust test for the presence of a microbe.For example, such a robust test can reduce the chances of afalse-positive or false-negative result in comparison with an assay thatrelies upon a single unique receptor to detect a given microbe. Further,this embodiment of the method can develop or employ working receptorsthat demonstrate higher binding affinity due to interaction withmultiple antigens or ligands on the same microbe (e.g., multivalentbinding).

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a bacterium. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the bacterium. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Thebacterium can exhibit characteristic binding to one or several of thecandidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sampleor characterizing or detecting the bacterium.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a virus particle. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the virus particle. The buildingblocks making up the artificial receptors can be naïve to the testligand. The virus particle can exhibit characteristic binding to one orseveral of the candidate artificial receptors from that array. The oneor several artificial receptors can be selected as an artificialreceptor (e.g., a working artificial receptor or a working artificialreceptor complex) that can be employed in methods for characterizing abiological sample or for characterizing or detecting the virus particle.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a biohazard. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the biohazard. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Thebiohazard can exhibit characteristic binding to one or several of thecandidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sample,or characterizing or detecting the biohazard.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting the Vibrio cholerae. The method caninclude evaluating an array including a significant number of candidateartificial receptors for binding to V. cholerae. The building blocksmaking up the artificial receptors can be naïve to the V. cholerae. TheV. cholerae can exhibit characteristic binding to one or several of thecandidate artificial receptors from that array. The one or severalartificial receptors can be selected as an artificial receptor (e.g., aworking artificial receptor or a working artificial receptor complex)that can be employed in methods for characterizing a biological sample,or characterizing or detecting V. cholerae.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a microbe. The method can includeevaluating an array including a significant number of candidateartificial receptors for binding to the microbe. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Oneor more artificial receptors that bind the microbe under appropriateconditions can be selected for use on an affinity support that can bindthat microbe. One or more artificial receptors that bind the microbe orcell sufficiently tightly under appropriate conditions can be selectedand its building blocks incorporated onto a scaffold molecule. Anembodiment of the method can employ a support with the one or moreartificial receptors or scaffold-receptors on its surface for binding orimmobilizing the microbe. A support with a plurality of artificialreceptors, each binding to a different portion of the microbe, on itssurface can be employed for multivalent capture or immobilization of themicrobe.

The present method can include selecting artificial receptors that binda particular microbe and/or the building blocks making up thesereceptors (e.g., bound to a scaffold molecule) as leads forpharmaceutical development or as active agents for modulating anactivity of the microbe or as an antibiotic against that microbe.

In an embodiment, the method can employ an array including a significantnumber of the present artificial receptors to produce an assay or systemfor characterizing or detecting a microbe of clinical or environmentalinterest. The method can include evaluating an array including asignificant number of candidate artificial receptors for binding to themicrobe of clinical or environmental interest. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Themicrobe of clinical or environmental interest can exhibit characteristicbinding to one or several of the candidate artificial receptors fromthat array. The one or several artificial receptors can be selected asan artificial receptor (e.g., a working artificial receptor or a workingartificial receptor complex) that can be employed in methods forcharacterizing a biological sample, or characterizing or detecting themicrobe of clinical or environmental interest.

Suitable microbes of clinical or environmental interest includebacteria, mycoplasma, fungus, rickettsia, or virus. Suitable bacteria ormycoplasma of clinical or environmental interest include Escherichiacoli (e.g., E. coli H157:07), Vibrio cholerae, Acinetobactercaicoaceticus, Haemophilus influenzae, Actinobacillus actinoides,Haemophilus parahaemolyticus, Actinobacillus lignieresii, Haemophilusparainfluenzae, Actinobacillus suis, Legionella pneumophila, Actinomycesbovis, Leptospira interrogans, Actinomyces israelli, Mima polymorphs,Aeromonas hydrophile, Moraxella lacunata, Arachnia propionica,Burkholderia mallei, Burkholderia pseudomallei, Moraxella osioensis,Arizona hinshawii, Mycobacterium osioensis, Bacillus cereus,Mycobacterium leprae, Bacteroides spp, Mycobacterium spp, Bartonellabacilliformis, Plesiomonas shigelloides, Bordetella bronchiseptica,Proteus spp, Clostridium difficile, Pseudomonas aeruginosa, Clostridiumsordellii, Salmonella cholerasuis, Clostridium tetani, Salmonellaenteritidis, Corynebacterium diphtheriae, Salmonella typhi, Edwardsiellatarda, Serratia marcescens, Enterobacter aerogenes, Shigella spp,Staphylococcus epidermidis, Francisella novicida, Vibrioparahaemolyticus, Haemophilus ducreyi, Haemophilus gallinarum,Haemophilus haemolyticus, Bacillus anthracis, Mycobacterium bovis,Bordetella pertussis, Mycobacterium tuberculosis, Borrella burgdorfii,Mycoplasma pneumoniae, Borrella spp, Neisseria gonorrhoeae,Campylobacter, Neisseria meningitides, Chlamydia psittaci, Nocardiaasteroids, Chlamydia trachomatis, Nocardia brasillensis, Clostridiumbotulinum, Pasteurella haemolytica, Clostridium chauvoei, Pasteureliamultocida, Clostridium haemolyticus, Pasteurella pneumotropica,Clostridium histolyticum, Pseudomonas pseudomallei, Clostridium novyl,Staphylococcus aureus, Clostridium perfringens, Streptobacillusmoniliformis, Clostridium septicum, Cyclospora cayatanensis,Streptococcus agalacetiae, Erysipelothrix insidiosa, Streptococcuspneumoniae, Klebsiella pneumoniae, Streptococcus pyogenes, Listeriamanocytogenes, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaenterocolitica, Brucella abortus, Brucella canis, Brucella melitensis,Brucella suis, and Francisella tularensis.

Suitable fungus include Absidia, Piedraia hortae, Aspergillus,Prototheca, Candida, Paecilomyces, Cryptococcus neoformans,Cryptosporidium parvum, Phialaphora, Dermatophilus congolensis,Rhizopus, Epidermophyton, Scopulariopsis, Exophiala, Sporothrixschenkii, Fusarium, Trichophyton, Madurella mycetomi, Toxoplasma,Trichosporon, Microsporum, Microsporidia, Wangiella dermatitidis, Mucor,Blastomyces dermatitidis, Giardia lamblia, Entamoeba histolytica,Coccidioides immitis, and Histoplasma capsulatum.

Suitable rickettsia or viruses of clinical or environmental interestinclude Coronaviruses, Hepatitis viruses, Hepatitis A virus,Myxo-Paramyxoviruses (Influenza viruses, Measles virus, Mumps virus,Newcastle disease virus), Picornavirus (Coxsackie viruses, Echoviruses,Poliomyelitis virus), Rickettsia akari, Rochalimaea Quintana,Rochalimaea vinsonii, Norwalk Agent, Adenoviruses, Arenaviruses(Lymphocytic choriomenigitis, Viscerotrophic strains), Herpesvirus Group(Herpesvirus hominis, Cytomegalovirus, Epstein-Barr virus,Caliciviruses, Pseudo-rabies virus, Varicella virus), HumanImmunodeficiency Virus, Parainfluenza viruses (Respiratory syncytialvirus, Subsclerosing panencephalitis virus), Picornaviruses(Poliomyelitis virus), Poxviruses Variola, Cowpox virus (Molluscumcontagiosum virus, Monkeypox virus, Orf virus, Paravaccinia virus,Tanapox virus, Vaccinia virus, Yabapox virus), Papovaviruses (SV 40virus, B-K-virus), Spongiform Encephalopathy Viruses (Creutzfeld-Jacobagent, Kuru agent, BSE), Rhabdoviruses (Rabies virus), Tobaviruses(Rubella virus), Coxiella burnetii, Rickettsia canada, Rickettsiaprowazekii, Rickettsia rickettsii, Rickettsia Tsutsugamushi, Rickettsiatyphi (R. mooseri), Spotted Fever Group Agents, Vesicular Stomatis Virus(VSV), and Toga, Arena (e.g., LCM, Junin, Lassa, Marchupo, Guanarito,etc.), Bunya (e.g., hantavirus, Rift Valley Fever, etc.), Flaviruses(Dengue), and Filoviruses (e.g., Ebola, Marburg, etc.) of all types,Nipah virus, viral encephalitis agents, LaCrosse, Kyasanur Forest virus,Yellow fever, and West Nile virus.

Suitable microbes of clinical or environmental interest include VariolaViruses, Congo-Crimean hemorrhagic fever, Tick-borne encephalitis viruscomplex (Absettarov, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease,Omsk hemorrhagic fever, and Russian Spring-Summer Encephalitis),Marburg, Ebola, Junin, Lassa, Machupo, Herpesvirus simiae, Bluetongue,Louping III, Rift Valley fever (Zing a), Wesselsbron, Foot and MouthDisease, Newcastle Disease, African Swine Fever, Vesicular exanthema,Swine vesicular disease, Rinderpest, African horse sickness, Avianinfluenza, and Sheep pox. Other components of interest include Ricinuscommunis.

Methods for Making and Using Affinity Supports

In an embodiment, a working artificial receptor or receptor complex canbe employed to produce or as an affinity support for any of the testligands described herein. For example, the present method can include amethod for producing an affinity support for a test ligand. This methodcan include selecting a working artificial receptor or receptor complexthat binds to the test ligand. This method can also include coupling theworking artificial receptor or receptor complex to a support. FIG. 20schematically illustrates an embodiment of such a method. The supportcan be suitable for use as an affinity support for, for example,chromatography, membrane filtration, electrophoresis (e.g., 1 or 2dimensional electrophoresis), or the like.

The present method can include selecting artificial receptors that binda particular test ligand and/or the building blocks making up thesereceptors (e.g., bound to a scaffold molecule) for isolation or analysisof a particular test ligand. The building blocks making up theartificial receptors can be naïve to the test ligand. For example, theartificial receptor can be employed as a receptor surface that can bindthe test ligand and remove (e.g., purify) it from a mixture orbiological sample.

Such a method can include contacting one or more candidate artificialreceptors with a test ligand of interest. The building blocks making upthe artificial receptors can be naïve to the test ligand. The method caninclude selecting one or more of the candidate artificial receptors thatbind the test ligand as working artificial receptor(s). The method canthen include employing the working artificial receptor(s) to make areceptor surface. Making a receptor surface can include coupling thebuilding blocks making up the working artificial receptor(s) to asupport. The support can have sufficient area to bind a significantquantity of the test ligand of interest. The support can be achromatography support or medium. The support can be a plate, tube, ormembrane. In an embodiment, binding of the test ligand of interest tothe support can be followed by eluting the test ligand of interest fromthe support. Eluting can employ a wash with a pH, buffer, solvent, saltconcentration, or ligand concentration effective to elute the testligand of interest from the support.

FIG. 21 schematically illustrates evaluating an array of candidateartificial receptors for binding of a test ligand and selecting one ormore working artificial receptors. The building blocks making up theartificial receptors can be naïve to the test ligand. FIG. 21illustrates that a receptor surface employing such a working artificialreceptor can be employed for binding a protein, immobilizing anantibody, binding a single enantiomer, or protecting a structuralfeature (e.g., a functional group) on a compound. In an embodiment, thereceptor surface can bind more than one structural feature on theprotein. In an embodiment, the working artificial receptor can beselected to bind the constant portion, rather than the variableportions, of an antibody. In an embodiment, the receptor surface caninclude a catalytic moiety that can catalyze a reaction of a functionalgroup of the bound test ligand. Such a catalytic moiety can be abuilding block, for example, an organometallic building block.

The present method can include selecting artificial receptors that binda particular isomer of a compound and/or the building blocks making upthese receptors (e.g., bound to a scaffold molecule) for isolation oranalysis of a particular isomer. For example, the artificial receptorcan be employed as a receptor surface that can bind the isomer andremove (e.g., purify) it from a mixture or biological sample.

Such a method can include contacting one or more candidate artificialreceptors with an isomer of interest. The building blocks making up theartificial receptors can be naïve to the test ligand. The method caninclude selecting one or more of the candidate artificial receptors thatbind the isomer as working artificial receptor(s). The method can theninclude employing the working artificial receptor(s) to make a receptorsurface. Making a receptor surface can include coupling the buildingblocks making up the working artificial receptor(s) to a support. Thesupport can have sufficient area to bind a significant quantity of theisomer of interest. The support can be a chromatography support ormedium. The support can be a plate, tube, or membrane. In an embodiment,binding of the isomer of interest to the support can be followed byeluting the isomer of interest from the support. Eluting can employ awash with a pH, buffer, solvent, salt concentration, or ligandconcentration effective to elute the isomer of interest from thesupport.

The present method can include selecting artificial receptors that bindor protect a particular structural feature of a compound and/or thebuilding blocks making up these receptors (e.g., bound to a scaffoldmolecule) for isolation or analysis of the compound including thestructural feature. For example, the artificial receptor can be employedas a receptor surface that can bind or protect the structural feature ofthe compound. Binding of the structural feature can be determined bylack of binding of an analogous compound the lacking the structuralfeature. Protection of the structural feature can be evaluated by thatstructural feature being unavailable to a, for example, solution phasereactive species when the compound is bound to the receptor surface.

Such a method can include contacting one or more candidate artificialreceptors with a compound of interest. The building blocks making up theartificial receptors can be naïve to the test ligand. The method caninclude selecting one or more of the candidate artificial receptors thatbind the structural feature of the compound as lead artificialreceptor(s). The lead artificial receptor can be evaluated forprotecting the structural feature. The method can then include employingthe working artificial receptor(s) to make a receptor surface. Making areceptor surface can include coupling the building blocks making up theworking artificial receptor(s) to a support. The support can havesufficient area to bind a significant quantity of the compound ofinterest. In an embodiment, binding of the compound of interest to thesupport can be followed by reacting a portion of the compound that isnot the bound or protected structure feature.

The present method can include selecting artificial receptors that binda particular peptide or protein and/or the building blocks making upthese receptors (e.g., bound to a scaffold molecule) for isolation oranalysis of a particular peptide or protein. For example, the artificialreceptor can be employed as a receptor surface that can bind the peptideor protein and remove (e.g., purify) it from a mixture or biologicalsample.

Such a method can include contacting one or more candidate artificialreceptors with a peptide or protein of interest. The building blocksmaking up the artificial receptors can be naïve to the test ligand. Themethod can include selecting one or more of the candidate artificialreceptors that bind the peptide or protein as working artificialreceptor(s). The method can then include employing the workingartificial receptor(s) to make a receptor surface. Making a receptorsurface can include coupling the building blocks making up the workingartificial receptor(s) to a support. The support can have sufficientarea to bind a significant quantity of the peptide or protein ofinterest. The support can be a chromatography support or medium. Thesupport can be a plate, tube, or membrane. In an embodiment, binding ofthe peptide or protein of interest to the support can be followed byeluting the peptide or protein of interest from the support. Eluting canemploy a wash with a pH, buffer, salt concentration, or ligandconcentration effective to elute the peptide or protein of interest fromthe support.

In an embodiment, the present artificial receptors can be employed toform selective membranes. Such a selective membrane can be based on amolecular gate including an artificial receptor surface. For example, anartificial receptor surface can line the walls of pores in the membraneand either allow or block a target molecule from passing through thepores. For example, an artificial receptor surface can line the walls ofpores in the membrane and act as “gatekeepers” on e.g. microcantilevers/molecular cantilevers to allow gate opening or closing onbinding of the target. The artificial receptors to be used in theselective membranes can be identified by exposing the target molecule toa plurality of distinct artificial receptors and then determining whichones it binds to. For example, the binding can be detected through anyof the techniques described herein, including fluorescence.

In certain embodiments, the method can include producing one or morereceptor surfaces, each receptor surface including building blocks froma working receptor for a particular test ligand. Such a method caninclude employing the receptor surface for chromatography of the testligand. Chromatographing the test ligand against a plurality of suchreceptor surfaces can rank the affinity of the surfaces for the testligand. Under a given set of conditions, the receptor surface thatretains the chromatographed test ligand the longest exhibits thegreatest affinity for the test ligand. The method can include selectingthe receptor surface with suitable (e.g., the greatest) affinity for useas an affinity support for the test ligand.

Any of a variety of supports can be employed as the affinity support. Incertain embodiments, the affinity support can be a dish, a tube, a well,a bead, a chromatography support, a microchannel, or the like. Theartificial receptor affinity support can be used in variousapplications, such as chromatography, microchannel devices, as animmunoassay support, or the like. A microchannel with the artificialreceptor on its surface can be employed as an analytical device. In anembodiment, the present artificial receptors can be employed to formbioactive surfaces. For example, receptor surfaces can be used tospecifically bind antibodies or enzymes.

Methods for Making and Using Reaction Supports

In an embodiment, a working artificial receptor or receptor complex canbe employed to produce or as a reaction support for any of the testligands described herein. For example, the present method can include amethod for producing a reaction support for at least one test ligand.This method can include selecting a working artificial receptor orreceptor complex that binds to the test ligand under conditions suitablefor a desired reaction with that test ligand. This method can alsoinclude coupling the working artificial receptor or receptor complex toa support. FIG. 20 schematically illustrates an embodiment of such amethod. The support can be suitable for use as a reaction support for,for example, oxidation, reduction, substitution, or displacementreactions.

The present method can include selecting artificial receptors that binda particular test ligand and/or the building blocks making up thesereceptors (e.g., bound to a scaffold molecule) for reaction of aparticular test ligand. For example, the artificial receptor can beemployed as a receptor surface that can bind the test ligand andposition it for reaction at a particular prochiral group, functionalgroup, or orientation.

Such a method can include contacting one or more candidate artificialreceptors with a test ligand of interest. The building blocks making upthe artificial receptors can be naïve to the test ligand. The method caninclude selecting one or more of the candidate artificial receptors thatbind the test ligand as working artificial receptor(s). The method canthen include employing the working artificial receptor(s) to make areceptor surface. Making a receptor surface can include coupling thebuilding blocks making up the working artificial receptor(s) to asupport. The support can have sufficient area to bind a desired quantityof the test ligand of interest. The support can be a chromatographysupport or medium. The support can be a plate, bead, tube, or membrane.

The method also includes contacting the support including bound testligand with a reactant for the desired reaction. Suitable reactantsinclude reducing agent, oxidizing agent, nucleophile, electrophile,solvent (e.g., aqueous or organic solvent), or the like. The method caninclude contacting with one or more reactants and selecting reactant orreactants suitable for participating in the desired reaction. Thisembodiment of the method includes reacting the test ligand with thereactant. In an embodiment, reacting can be followed by washing thereactant or side products from the support. In an embodiment, reactingcan be followed by eluting the product (e.g., reacted test ligand) fromthe support. Eluting can employ a wash with a pH, buffer, solvent, saltconcentration, or ligand concentration effective to elute the productfrom the support.

FIG. 21 schematically illustrates evaluating an array of candidateartificial receptors for binding of a test ligand and selecting one ormore working artificial receptors. The building blocks making up theartificial receptors can be naïve to the test ligand. FIG. 21illustrates that a receptor surface employing such a working artificialreceptor can be employed for binding a test ligand. The test ligand canbe bound in an orientation that leaves a reactive moiety available forreaction with a reactant placed into contact with the receptor surface.In an embodiment, the test ligand can be bound in an orientation thatoccludes or protects a second reactive moiety from reacting with thereactant. This embodiment includes reacting the test ligand and releaseof the reacted test ligand from the receptor surface. Specifically, theillustration shows the reduction of an aldehyde with sodium borohydrideto produce an alcohol. In an embodiment, the receptor surface caninclude a catalytic moiety that can catalyze a reaction of a functionalgroup of the bound to test ligand the catalytic reaction can also employthe reactant. Such a catalytic moiety can be a building block, forexample, an organometallic building block.

The present method can include selecting artificial receptors that bindor protect a particular structural feature of a compound and/or thebuilding blocks making up these receptors (e.g., bound to a scaffoldmolecule) for reacting the compound including the structural feature.For example, the artificial receptor can be employed as a receptorsurface that can bind and protect the structural feature of the compoundwhile another feature of the compound reacts with a reactant. Protectionof the structural feature can be evaluated by that structural featurenot reacting with the reactant. For example, a substrate (e.g. asteroid) can be stereospecifically bound to the artificial receptor andpresent a particular moiety/sub-structure/“face” for reaction with areagent in solution.

In an embodiment, a first side of a molecule (or a functional group) isbound to a receptor surface while a second side is left exposed. Then areagent is added that could react with either side (or group) but ishindered from reacting with the first side of the molecule since it isbound to the receptor surface, accordingly the reagent reacts with thesecond side of the molecule only.

Such a method can include contacting one or more candidate artificialreceptors with a compound of interest. The method can include selectingone or more of the candidate artificial receptors that bind thestructural feature of the compound as lead artificial receptor(s). Thelead artificial receptor can be evaluated for protecting the structuralfeature. The method can then include employing the working artificialreceptor(s) to make a receptor surface. Making a receptor surface caninclude coupling the building blocks making up the working artificialreceptor(s) to a support. The support can have sufficient area to bind adesired quantity of the compound of interest. In an embodiment, bindingof the compound of interest to the support can be followed by reacting aportion of the compound that is not the bound or protected structurefeature.

Conventional synthesis of a chiral compound generally requirescomplicated procedures. In an embodiment, the present candidateartificial receptors can be employed to find receptor surfaces thatprovide a spatially oriented binding surface for a stereospecificreaction. For example, an artificial receptor surface can bind a smallmolecule so that particular functional groups are exposed to theenvironment, and others are obscured by the receptor. In this manner,the stereospecificity of the reaction can be controlled. Therefore, anartificial receptor surface can be employed in synthesis includingchiral induction. Similarly, regiospecificity can also be controlledusing receptors of the present invention.

The present method can include selecting artificial receptors that binda first reaction ligand and a second reaction ligand or the buildingblocks making up these receptors (e.g., bound to a scaffold molecule)for a reaction including the first and second reaction ligands. Forexample, the artificial receptor can be employed as a receptor surfacethat can bind the first reaction ligand and the second reaction ligandat a distance or orientation at which these ligands can react with oneanother. The reaction can optionally include one or more reactants notbound to the receptor surface.

Such a method can include contacting one or more candidate artificialreceptors with a first reaction ligand and a second reaction ligand. Thebuilding blocks making up the artificial receptors can be naïve to theligands. The method can include selecting one or more of the candidateartificial receptors that bind both of the reaction ligands as workingartificial receptor(s). The method can then include employing theworking artificial receptor(s) to make a receptor surface. Making areceptor surface can include coupling the building blocks making up theworking artificial receptor(s) to a support. The support can havesufficient area to bind a desired quantity of the first and secondreaction ligands. The support can be a chromatography support or medium.The support can be a plate, bead, tube, or membrane. The first andsecond reaction ligands can be bound to the support at one or severalmolar ratios, the reaction evaluated, and a molar ratio selected forconducting the reaction.

The method can also include contacting the support including boundreaction ligands with a reactant for the desired reaction. Suitablereactants include reducing agent, oxidizing agent, nucleophile,electrophile, or the like. This embodiment of the method includesreacting the test ligand with the reactant. In an embodiment, reactingcan be followed by washing the reactant or side products from thesupport. In an embodiment, the first and second reaction ligands reactwithout the reactant. In an embodiment, reacting can be followed byeluting the product (e.g., reacted test ligand) from the support.Eluting can employ a wash with a pH, buffer, solvent, saltconcentration, or ligand concentration effective to elute the productfrom the support.

In an embodiment, the present candidate artificial receptors can beemployed to find receptor surfaces that provide a spatially orientedbinding surface for a stereospecific reaction. For example, anartificial receptor surface can bind a small molecule with particularfunctional groups exposed to the environment, and others obscured by thereceptor. Such an artificial receptor surface can be employed insynthesis including chiral induction. For example, a substrate (e.g. asteroid) can be stereospecifically bound to the artificial receptor andpresent a particular moiety/sub-structure/“face” for reaction with areagent in solution. Similarly, the artificial receptor surface can actas a protecting group where a reactive moiety of a molecule is“protected” by binding to the receptor surface so that a differentmoiety with similar reactivity can be transformed.

In an embodiment, the one or more working artificial receptors that binda plurality (e.g., 2) of the reactants can be produced on a substrateand the reactants bound. Each receptor with a plurality of boundreactants can then be screened against one or more reagents orconditions (e.g., various molar ratios of the reactants or varioussolvents). The artificial receptor allowing or promoting reactionbetween the two or more reactants can be identified. The artificialreceptor can then be produced on a substrate to provide a reactor forthe reaction of interest.

Any of a variety of supports can be employed as the reaction support. Incertain embodiments, the reaction support can be a dish, a tube, a well,a bead, a chromatography support, a microchannel, or the like. Theartificial receptor reaction support can be used in variousapplications, such as a microchannel device. A microchannel with theartificial receptor on its surface can be employed as a reactor.

Methods of Making an Artificial Receptor

The present invention relates to a method of making an artificialreceptor or a candidate artificial receptor. In an embodiment, thismethod includes preparing a spot or region on a support, the spot orregion including a plurality of building blocks immobilized on thesupport. The method can include forming a plurality of spots on a solidsupport, each spot including a plurality of building blocks, andimmobilizing (e.g., reversibly) a plurality of building blocks on thesolid support in each spot. In an embodiment, an array of such spots isreferred to as a heterogeneous building block array.

The method can include mixing a plurality of building blocks andemploying the mixture in forming the spot(s). Alternatively, the methodcan include spotting individual building blocks on the support. Couplingbuilding blocks to the support can employ covalent bonding ornoncovalent interactions. Suitable noncovalent interactions includeinteractions between ions, hydrogen bonding, van der Waals interactions,and the like. In an embodiment, the support can be functionalized withmoieties that can engage in covalent bonding or noncovalentinteractions. Forming spots can yield a microarray of spots ofheterogeneous combinations of building blocks, each of which can be acandidate artificial receptor. The method can apply or spot buildingblocks onto a support in combinations of 2, 3, 4, or more buildingblocks.

In an embodiment, the present method can be employed to produce a solidsupport having on its surface a plurality of regions or spots, eachregion or spot including a plurality of building blocks. For example,the method can include spotting a glass slide with a plurality of spots,each spot including a plurality of building blocks. Such a spot can bereferred to as including heterogeneous building blocks. A plurality ofspots of building blocks can be referred to as an array of spots.

In an embodiment, the present method includes making a receptor surface.Making a receptor surface can include forming a region on a solidsupport, the region including a plurality of building blocks, andimmobilizing (e.g., reversibly) the plurality of building blocks to thesolid support in the region. The method can include mixing a pluralityof building blocks and employing the mixture in forming the region orregions. Alternatively, the method can include applying individualbuilding blocks in a region on the support. Forming a region on asupport can be accomplished, for example, by soaking a portion of thesupport with the building block solution. The resulting coatingincluding building blocks can be referred to as including heterogeneousbuilding blocks.

A region including a plurality of building blocks can be independent anddistinct from other regions including a plurality of building blocks. Inan embodiment, one or more regions including a plurality of buildingblocks can overlap to produce a region including the combinedpluralities of building blocks. In an embodiment, two or more regionsincluding a single building block can overlap to form one or moreregions each including a plurality of building blocks. The overlappingregions can be envisioned, for example, as portions of overlap in a Vendiagram, or as portions of overlap in a pattern like a plaid or tweed.

In an embodiment, the method produces a spot or surface with a densityof building blocks sufficient to provide interactions of more than onebuilding block with a ligand. That is, the building blocks can be inproximity to one another. Proximity of different building blocks can bedetected by determining different (e.g., greater) binding of a testligand to a spot or surface including a plurality of building blockscompared to a spot or surface including only one of the building blocks.

In an embodiment, the method includes forming an array of heterogeneousspots made from combinations of a subset of the total building blocksand/or smaller groups of the building blocks in each spot. That is, themethod forms spots including only, for example, 2 or 3 building blocks,rather than 4 or 5. For example, the method can form spots fromcombinations of a full set of building blocks (e.g. 81 of a set of 81)in groups of 2 and/or 3. For example, the method can form spots fromcombinations of a subset of the building blocks (e.g., 25 of the set of81) in groups of 4 or 5. For example, the method can form spots fromcombinations of a subset of the building blocks (e.g., 25 of a set of81) in groups of 2 or 3. The method can include forming additionalarrays incorporating building blocks, lead artificial receptors, orstructurally similar building blocks.

In an embodiment, the method includes forming an array including one ormore spots that function as controls for validating or evaluatingbinding to artificial receptors of the present invention. In anembodiment, the method includes forming one or more regions, tubes, orwells that function as controls for validating or evaluating binding toartificial receptors of the present invention. Such a control spot,region, tube, or well can include no building block, only a singlebuilding block, only functionalized lawn, or combinations thereof.

The method can immobilize (e.g., reversibly) building blocks on supportsusing known methods for immobilizing compounds of the types employed asbuilding blocks. Coupling building blocks to the support can employcovalent bonding or noncovalent interactions. Suitable noncovalentinteractions include interactions between ions, hydrogen bonding, vander Waals interactions, and the like. In an embodiment, the support canbe functionalized with moieties that can engage in reversible covalentbonding, moieties that can engage in noncovalent interactions, a mixtureof these moieties, or the like.

In an embodiment, the support can be functionalized with moieties thatcan engage in covalent bonding, e.g., reversible covalent bonding. Thepresent invention can employ any of a variety of the numerous knownfunctional groups, reagents, and reactions for forming reversiblecovalent bonds. Suitable reagents for forming reversible covalent bondsinclude those described in Green, T W; Wuts, P G M (1999), ProtectiveGroups in Organic Synthesis Third Edition, Wiley-Interscience, New York,779 pp. For example, the support can include functional groups such as acarbonyl group, a carboxyl group, a silane group, boric acid or ester,an amine group (e.g., a primary, secondary, or tertiary amine, ahydroxylamine, a hydrazine, or the like), a thiol group, an alcoholgroup (e.g., primary, secondary, or tertiary alcohol), a diol group(e.g., a 1,2 diol or a 1,3 diol), a phenol group, a catechol group, orthe like. These functional groups can form groups with reversiblecovalent bonds, such as ether (e.g., alkyl ether, silyl ether,thioether, or the like), ester (e.g., alkyl ester, phenol ester, cyclicester, thioester, or the like), acetal (e.g., cyclic acetal), ketal(e.g., cyclic ketal), silyl derivative (e.g., silyl ether), boronate(e.g., cyclic boronate), amide, hydrazide, imine, carbamate, or thelike. Such a functional group can be referred to as a covalent bondingmoiety, e.g., a first covalent bonding moiety.

A carbonyl group on the support and an amine group on a building blockcan form an imine or Schiff's base. The same is true of an amine groupon the support and a carbonyl group on a building block. A carbonylgroup on the support and an alcohol group on a building block can forman acetal or ketal. The same is true of an alcohol group on the supportand a carbonyl group on a building block. A thiol (e.g., a first thiol)on the support and a thiol (e.g., a second thiol) on the building blockcan form a disulfide.

A carboxyl group on the support and an alcohol group on a building blockcan form an ester. The same is true of an alcohol group on the supportand a carboxyl group on a building block. Any of a variety of alcoholsand carboxylic acids can form esters that provide covalent bonding thatcan be reversed in the context of the present invention. For example,reversible ester linkages can be formed from alcohols such as phenolswith electron withdrawing groups on the aryl ring, other alcohols withelectron withdrawing groups acting on the hydroxyl-bearing carbon, otheralcohols, or the like; and/or carboxyl groups such as those withelectron withdrawing groups acting on the acyl carbon (e.g.,nitrobenzylic acid, R—CF₂—COOH, R—CCl₂—COOH, and the like), othercarboxylic acids, or the like.

In an embodiment, the support, matrix, or lawn can be functionalizedwith moieties that can engage in noncovalent interactions. For example,the support can include functional groups such as an ionic group, agroup that can hydrogen bond, or a group that can engage in van derWaals or other hydrophobic interactions. Such functional groups caninclude cationic groups, anionic groups, lipophilic groups, amphiphilicgroups, and the like.

In an embodiment, the support, matrix, or lawn includes a charged moiety(e.g., a first charged moiety). Suitable charged moieties includepositively charged moieties and negatively charged moieties. Suitablepositively charged moieties (e.g., at neutral pH in aqueouscompositions) include amines, quaternary ammonium moieties, ferrocene,or the like. Suitable negatively charged moieties (e.g., at neutral pHin aqueous compositions) include carboxylates, phenols substituted withstrongly electron withdrawing groups (e.g., tetrachlorophenols),phosphates, phosphonates, phosphinates, sulphates, sulphonates,thiocarboxylates, hydroxamic acids, or the like.

In an embodiment, the support, matrix, or lawn includes groups that canhydrogen bond (e.g., a first hydrogen bonding group), either as donorsor acceptors. The support, matrix, or lawn can include a surface orregion with groups that can hydrogen bond. For example, the support,matrix, or lawn can include a surface or region including one or morecarboxyl groups, amine groups, hydroxyl groups, carbonyl groups, or thelike. Ionic groups can also participate in hydrogen bonding.

In an embodiment, the support, matrix, or lawn includes a lipophilicmoiety (e.g., a first lipophilic moiety). Suitable lipophilic moietiesinclude branched or straight chain C₆₋₃₆ alkyl, C₈₋₂₄ alkyl, C₁₂₋₂₄alkyl, C₁₂₋₁₈ alkyl, or the like; C₆₋₃₆ alkenyl, C₈₋₂₄ alkenyl, C₁₂₋₂₄alkenyl, C₁₂₋₁₈ alkenyl, or the like, with, for example, 1 to 4 doublebonds; C₆₋₃₆ alkynyl, C₈₋₂₄ alkynyl, C₁₂₋₂₄ alkynyl, C₁₂₋₁₈ alkynyl, orthe like, with, for example, 1 to 4 triple bonds; chains with 1-4 doubleor triple bonds; chains including aryl or substituted aryl moieties(e.g., phenyl or naphthyl moieties at the end or middle of a chain);polyaromatic hydrocarbon moieties; cycloalkane or substituted alkanemoieties with numbers of carbons as described for chains; combinationsor mixtures thereof; or the like. The alkyl, alkenyl, or alkynyl groupcan include branching; within chain functionality like an ether group;terminal functionality like alcohol, amide, carboxylate or the like; orthe like. A lipophilic moiety like a quaternary ammonium lipophilicmoiety can also include a positive charge.

Artificial Receptors

A candidate artificial receptor, a lead artificial receptor, or aworking artificial receptor includes combination of building blocksimmobilized (e.g., reversibly) on, for example, a support. An individualartificial receptor can be a heterogeneous building block spot on aslide or a plurality of building blocks coated on a slide, tube, orwell. The building blocks can be immobilized through any of a variety ofinteractions, such as covalent, electrostatic, or hydrophobicinteractions. For example, the building block and support or lawn caneach include one or more functional groups or moieties that can formcovalent, electrostatic, hydrogen bonding, van der Waals, or likeinteractions.

An array of candidate artificial receptors can be a commercial productsold to parties interested in using the candidate artificial receptorsas implements in developing receptors for test ligands of interest. Inan embodiment, a useful array of candidate artificial receptors includesat least one glass slide, the at least one glass slide including spotsof a predetermined number of combinations of members of a set ofbuilding blocks, each combination including a predetermined number ofbuilding blocks.

One or more lead artificial receptors can be developed from a pluralityof candidate artificial receptors. In an embodiment, a lead artificialreceptor includes a combination of building blocks and binds detectablequantities of test ligand upon exposure to, for example, severalpicomoles of test ligand at a concentration of 1, 0.1, or 0.01 μg/ml, orat 1, 0.1, or 0.01 ng/ml test ligand; at a concentration of 0.01 μg/ml,or at 1, 0.1, or 0.01 ng/ml test ligand; or a concentration of 1, 0.1,or 0.01 ng/ml test ligand.

Artificial receptors, particularly candidate or lead artificialreceptors, can be in the form of an array of artificial receptors. Suchan array can include, for example, 1.66 million spots, each spotincluding one combination of 4 building blocks from a set of 81 buildingblocks. Such an array can include, for example, 28,000 spots, each spotincluding one combination of 2, 3, or 4 building blocks from a set of 29building blocks. Each spot is a candidate artificial receptor and acombination of building blocks. The array can also be constructed toinclude lead artificial receptors. For example, the array of artificialreceptors can include combinations of fewer building blocks and/or asubset of the building blocks.

In an embodiment, an array of candidate artificial receptors includesbuilding blocks of general Formula 2 (shown hereinbelow), with RE₁ beingB1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9 (shown hereinbelow) and withRE₂ being A1, A2, A3, A3a, A4, A5, A6, A7, A8, or A9 (shownhereinbelow). In an embodiment, the framework is tyrosine.

One or more working artificial receptors can be developed from one ormore lead artificial receptors. In an embodiment, a working artificialreceptor includes a combination of building blocks and bindscategorizing or identifying quantities of test ligand upon exposure to,for example, several picomoles of test ligand at a concentration of 100,10, 1, 0.1, 0.01, or 0.001 ng/ml test ligand; at a concentration of 10,1, 0.1, 0.01, or 0.001 ng/ml test ligand; or a concentration of 1, 0.1,0.01, or 0.001 ng/ml test ligand.

In an embodiment, the artificial receptor of the invention includes aplurality of building blocks coupled to a support. In an embodiment, theplurality of building blocks can include or be building blocks ofFormula 2 (shown below). An abbreviation for the building blockincluding a linker, a tyrosine framework, and recognition elements AxByis TyrAxBy. In an embodiment, a candidate artificial receptor caninclude combinations of building blocks of formula TyrA1B1, TyrA2B2,TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3, TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8,TyrA5B5, TyrA6B2, TyrA6B4, TyrA6B6, TyrA6B8, TyrA7B7, TyrA8B2, TyrA8B4,TyrA8B6, or TyrA8B8.

The present artificial receptors can employ any of a variety of supportsto which building blocks or other array materials can be coupled. Forexample, the support can be glass or plastic; a slide, a tube, or awell; an optical fiber; a nanotube or a buckyball, a nanodevice; adendrimer, or a scaffold; or the like.

The present artificial receptors can include a signal element thatproduces a detectable signal when a test ligand is bound to thereceptor. In an embodiment, the signal element can produce an opticalsignal or a electrochemical signal. Suitable optical signals includechemiluminescence or fluorescence. The signal element can be afluorescent moiety. The fluorescent molecule can be one that is quenchedby binding to the artificial receptor. For example, the signal elementcan be a molecule that fluoresces only when binding occurs. Suitableelectrochemical signal elements include those that give rise to currentor a potential. Suitable electrochemical signal elements include phenolsand anilines, such as those with substitutents oriented ortho or para toone another, polynuclear aromatic hydrocarbons, sulfide-disulfide,sulfide-sulfoxide-sulfone, polyenes, polyeneynes, and the like. Suitableelectrochemical signal elements include quinones and ferrocenes.

Building Blocks

The present invention relates to building blocks for making or formingcandidate artificial receptors. Building blocks can be designed, made,and selected to provide a variety of structural characteristics among asmall number of compounds. A building block can provide one or morestructural characteristics such as positive charge, negative charge,acid, base, electron acceptor, electron donor, hydrogen bond donor,hydrogen bond acceptor, free electron pair, π electrons, chargepolarization, hydrophilicity, hydrophobicity, and the like. A buildingblock can be bulky or it can be small.

A building block can be visualized as including several components, suchas one or more frameworks, one or more linkers, and/or one or morerecognition elements. The framework can be covalently coupled to each ofthe other building block components. The linker can be covalentlycoupled to the framework. The linker can be coupled to a support throughone or more of covalent, electrostatic, hydrogen bonding, van der Waals,or like interactions. The recognition element can be covalently coupledto the framework. In an embodiment, a building block includes aframework, a linker, and a recognition element. In an embodiment, abuilding block includes a framework, a linker, and two recognitionelements.

A description of general and specific features and functions of avariety of building blocks and their synthesis can be found in copendingU.S. patent application Ser. Nos. 10/244,727, filed Sep. 16, 2002,10/813,568, filed Mar. 29, 2004, and Application No. PCT/US03/05328,filed Feb. 19, 2003, each entitled “ARTIFICIAL RECEPTORS, BUILDINGBLOCKS, AND METHODS”; U.S. patent application Ser. Nos. 10/812,850 and10/813,612, and application No. PCT/US2004/009649, each filed Mar. 29,2004 and each entitled “ARTIFICIAL RECEPTORS INCLUDING REVERSIBLYIMMOBILIZED BUILDING BLOCKS, THE BUILDING BLOCKS, AND METHODS”; and U.S.Provisional Patent Application Nos. 60/499,965, filed Sep. 3, 2003, and60/526,699, filed Dec. 2, 2003, each entitled BUILDING BLOCKS FORARTIFICIAL RECEPTORS; the disclosures of which are incorporated hereinby reference. These patent documents include, in particular, a detailedwritten description of: function, structure, and configuration ofbuilding blocks, framework moieties, recognition elements, synthesis ofbuilding blocks, specific embodiments of building blocks, specificembodiments of recognition elements, and sets of building blocks.

Framework

The framework can be selected for functional groups that provide forcoupling to the recognition moiety and for coupling to or being thelinking moiety. The framework can interact with the ligand as part ofthe artificial receptor. In an embodiment, the framework includesmultiple reaction sites with orthogonal and reliable functional groupsand with controlled stereochemistry. Suitable functional groups withorthogonal and reliable chemistries include, for example, carboxyl,amine, hydroxyl, phenol, carbonyl, and thiol groups, which can beindividually protected, deprotected, and derivatized. In an embodiment,the framework has two, three, or four functional groups with orthogonaland reliable chemistries. In an embodiment, the framework has threefunctional groups. In such an embodiment, the three functional groupscan be independently selected, for example, from carboxyl, amine,hydroxyl, phenol, carbonyl, or thiol group. The framework can includealkyl, substituted alkyl, cycloalkyl, heterocyclic, substitutedheterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, and likemoieties.

A general structure for a framework with three functional groups can berepresented by Formula 1a:

A general structure for a framework with four functional groups can berepresented by Formula 1b:

In these general structures: R₁ can be a 1-12, a 1-6, or a 1-4 carbonalkyl, substituted alkyl, cycloalkyl, heterocyclic, substitutedheterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, or likegroup; and F₁, F₂, F₃, or F₄ can independently be a carboxyl, amine,hydroxyl, phenol, carbonyl, or thiol group. F₁, F₂, F₃, or F₄ canindependently be a 1-12, a 1-6, a 1-4 carbon alkyl, substituted alkyl,cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl, aryl,heteroaryl, heteroaryl alkyl, or inorganic group substituted withcarboxyl, amine, hydroxyl, phenol, carbonyl, or thiol group. F₃ and/orF₄ can be absent.

A variety of compounds fit the formulas and text describing theframework including amino acids, and naturally occurring or syntheticcompounds including, for example, oxygen and sulfur functional groups.The compounds can be racemic, optically active, or achiral. For example,the compounds can be natural or synthetic amino acids, α-hydroxy acids,thioic acids, and the like.

Suitable molecules for use as a framework include a natural or syntheticamino acid, particularly an amino acid with a functional group (e.g.,third functional group) on its side chain. Amino acids include carboxyland amine functional groups. The side chain functional group caninclude, for natural amino acids, an amine (e.g., alkyl amine,heteroaryl amine), hydroxyl, phenol, carboxyl, thiol, thioether, oramidino group. Natural amino acids suitable for use as frameworksinclude, for example, serine, threonine, tyrosine, aspartic acid,glutamic acid, asparagine, glutamine, cysteine, lysine, arginine,histidine. Synthetic amino acids can include the naturally occurringside chain functional groups or synthetic side chain functional groupswhich modify or extend the natural amino acids with alkyl, substitutedalkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,aryl, heteroaryl, heteroaryl alkyl, and like moieties as framework andwith carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol functionalgroups. Suitable synthetic amino acids include β-amino acids and homo orβ analogs of natural amino acids. In an embodiment, the framework aminoacid can be serine, threonine, or tyrosine, e.g., serine or tyrosine,e.g., tyrosine.

Although not limiting to the present invention, a framework amino acid,such as serine, threonine, or tyrosine, with a linker and tworecognition elements can be visualized with one of the recognitionelements in a pendant orientation and the other in an equatorialorientation, relative to the extended carbon chain of the framework.

All of the naturally occurring and many synthetic amino acids arecommercially available. Further, forms of these amino acids derivatizedor protected to be suitable for reactions for coupling to recognitionelement(s) and/or linkers can be purchased or made by known methods(see, e.g., Green, T W; Wuts, P G M (1999), Protective Groups in OrganicSynthesis Third Edition, Wiley-Interscience, New York, 779 pp.;Bodanszky, M.; Bodanszky, A. (1994), The Practice of Peptide SynthesisSecond Edition, Springer-Verlag, New York, 217 pp.).

Recognition Element

The recognition element can be selected to provide one or morestructural characteristics to the building block. The recognitionelement can interact with the ligand as part of the artificial receptor.For example, the recognition element can provide one or more structuralcharacteristics such as positive charge, negative charge, acid, base,electron acceptor, electron donor, hydrogen bond donor, hydrogen bondacceptor, free electron pair, π electrons, charge polarization,hydrophilicity, hydrophobicity, and the like. A recognition element canbe a small group or it can be bulky.

In an embodiment the recognition element can be a 1-12, a 1-6, or a 1-4carbon alkyl, substituted alkyl, cycloalkyl, heterocyclic, substitutedheterocyclic, aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, or likegroup. The recognition element can be substituted with a group thatincludes or imparts positive charge, negative charge, acid, base,electron acceptor, electron donor, hydrogen bond donor, hydrogen bondacceptor, free electron pair, π electrons, charge polarization,hydrophilicity, hydrophobicity, and the like.

Recognition elements with a positive charge (e.g., at neutral pH inaqueous compositions) include amines, quaternary ammonium moieties,sulfonium, phosphonium, ferrocene, and the like. Suitable amines includealkyl amines, alkyl diamines, heteroalkyl amines, aryl amines,heteroaryl amines, aryl alkyl amines, pyridines, heterocyclic amines(saturated or unsaturated, the nitrogen in the ring or not), amidines,hydrazines, and the like. Alkyl amines generally have 1 to 12 carbons,e.g., 1-8, and rings can have 3-12 carbons, e.g., 3-8. Suitable alkylamines include that of formula B9. Suitable heterocyclic or alkylheterocyclic amines include that of formula A9. Suitable pyridinesinclude those of formulas A5 and B5. Any of the amines can be employedas a quaternary ammonium compound. Additional suitable quaternaryammonium moieties include trimethyl alkyl quaternary ammonium moieties,dimethyl ethyl alkyl quaternary ammonium moieties, dimethyl alkylquaternary ammonium moieties, aryl alkyl quaternary ammonium moieties,pyridinium quaternary ammonium moieties, and the like.

Recognition elements with a negative charge (e.g., at neutral pH inaqueous compositions) include carboxylates, phenols substituted withstrongly electron withdrawing groups (e.g., substitutedtetrachlorophenols), phosphates, phosphonates, phosphinates, sulphates,sulphonates, thiocarboxylates, and hydroxamic acids. Suitablecarboxylates include alkyl carboxylates, aryl carboxylates, and arylalkyl carboxylates. Suitable phosphates include phosphate mono-, di-,and tri-esters, and phosphate mono-, di-, and tri-amides. Suitablephosphonates include phosphonate mono- and di-esters, and phosphonatemono- and di-amides (e.g., phosphonamides). Suitable phosphinatesinclude phosphinate esters and amides.

Recognition elements with a negative charge and a positive charge (atneutral pH in aqueous compositions) include sulfoxides, betaines, andamine oxides.

Acidic recognition elements can include carboxylates, phosphates,sulphates, and phenols. Suitable acidic carboxylates includethiocarboxylates. Suitable acidic phosphates include the phosphateslisted hereinabove.

Basic recognition elements include amines. Suitable basic amines includealkyl amines, aryl amines, aryl alkyl amines, pyridines, heterocyclicamines (saturated or unsaturated, the nitrogen in the ring or not),amidines, and any additional amines listed hereinabove. Suitable alkylamines include that of formula B9. Suitable heterocyclic or alkylheterocyclic amines include that of formula A9. Suitable pyridinesinclude those of formulas A5 and B5.

Recognition elements including a hydrogen bond donor include amines,amides, carboxyls, protonated phosphates, protonated phosphonates,protonated phosphinates, protonated sulphates, protonated sulphinates,alcohols, and thiols. Suitable amines include alkyl amines, aryl amines,aryl alkyl amines, pyridines, heterocyclic amines (saturated orunsaturated, the nitrogen in the ring or not), amidines, ureas, and anyother amines listed hereinabove. Suitable alkyl amines include that offormula B9. Suitable heterocyclic or alkyl heterocyclic amines includethat of formula A9. Suitable pyridines include those of formulas A5 andB5. Suitable protonated carboxylates, protonated phosphates includethose listed hereinabove. Suitable amides include those of formulas A8and B8. Suitable alcohols include primary alcohols, secondary alcohols,tertiary alcohols, and aromatic alcohols (e.g., phenols). Suitablealcohols include those of formulas A7 (a primary alcohol) and B7 (asecondary alcohol).

Recognition elements including a hydrogen bond acceptor or one or morefree electron pairs include amines, amides, carboxylates, carboxylgroups, phosphates, phosphonates, phosphinates, sulphates, sulphonates,alcohols, ethers, thiols, and thioethers. Suitable amines include alkylamines, aryl amines, aryl alkyl amines, pyridines, heterocyclic amines(saturated or unsaturated, the nitrogen in the ring or not), amidines,ureas, and amines as listed hereinabove. Suitable alkyl amines includethat of formula B9. Suitable heterocyclic or alkyl heterocyclic aminesinclude that of formula A9. Suitable pyridines include those of formulasA5 and B5. Suitable carboxylates include those listed hereinabove.Suitable amides include those of formulas A8 and B8. Suitablephosphates, phosphonates and phosphinates include those listedhereinabove. Suitable alcohols include primary alcohols, secondaryalcohols, tertiary alcohols, aromatic alcohols, and those listedhereinabove. Suitable alcohols include those of formulas A7 (a primaryalcohol) and B7 (a secondary alcohol). Suitable ethers include alkylethers, aryl alkyl ethers. Suitable alkyl ethers include that of formulaA6. Suitable aryl alkyl ethers include that of formula A4. Suitablethioethers include that of formula B6.

Recognition elements including uncharged polar or hydrophilic groupsinclude amides, alcohols, ethers, thiols, thioethers, esters, thioesters, boranes, borates, and metal complexes. Suitable amides includethose of formulas A8 and B8. Suitable alcohols include primary alcohols,secondary alcohols, tertiary alcohols, aromatic alcohols, and thoselisted hereinabove. Suitable alcohols include those of formulas A7 (aprimary alcohol) and B7 (a secondary alcohol). Suitable ethers includethose listed hereinabove. Suitable ethers include that of formula A6.Suitable aryl alkyl ethers include that of formula A4.

Recognition elements including uncharged hydrophobic groups includealkyl (substituted and unsubstituted), alkene (conjugated andunconjugated), alkyne (conjugated and unconjugated), aromatic. Suitablealkyl groups include lower alkyl, substituted alkyl, cycloalkyl, arylalkyl, and heteroaryl alkyl. Suitable lower alkyl groups include thoseof formulas A1, A3, A3a, and B1. Suitable aryl alkyl groups includethose of formulas A3, A3a, A4, B3, B3a, and B4. Suitable alkylcycloalkyl groups include that of formula B2. Suitable alkene groupsinclude lower alkene and aryl alkene. Suitable aryl alkene groupsinclude that of formula B4. Suitable aromatic groups includeunsubstituted aryl, heteroaryl, substituted aryl, aryl alkyl, heteroarylalkyl, alkyl substituted aryl, and polyaromatic hydrocarbons. Suitablearyl alkyl groups include those of formulas A3, A3a and B4. Suitablealkyl heteroaryl groups include those of formulas A5 and B5.

Spacer (e.g., small) recognition elements include hydrogen, methyl,ethyl, and the like. Bulky recognition elements include 7 or more carbonor hetero atoms.

Formulas A1-A9 and B1-B9 are:

These A and B recognition elements can be called derivatives of,according to a standard reference: A1, ethylamine; A2, isobutylamine;A3, phenethylamine; A4, 4-methoxyphenethylamine;A5,2-(2-aminoethyl)pyridine; A6,2-methoxyethylamine; A7, ethanolamine;A8, N-acetylethylenediamine; A9,1-(2-aminoethyl)pyrrolidine; B1, aceticacid, B2, cyclopentylpropionic acid; B3,3-chlorophenylacetic acid; B4,cinnamic acid; B5, 3-pyridinepropionic acid; B6, (methylthio)aceticacid; B7,3-hydroxybutyric acid; B8, succinamic acid; andB9,4-(dimethylamino)butyric acid.

In an embodiment, the recognition elements include one or more of thestructures represented by formulas A1, A2, A3, A3a, A4, A5, A6, A7, A8,and/or A9 (the A recognition elements) and/or B1, B2, B3, B3a, B4, B5,B6, B7, B8, and/or B9 (the B recognition elements). In an embodiment,each building block includes an A recognition element and a Brecognition element. In an embodiment, a group of 81 such buildingblocks includes each of the 81 unique combinations of an A recognitionelement and a B recognition element. In an embodiment, the A recognitionelements are linked to a framework at a pendant position. In anembodiment, the B recognition elements are linked to a framework at anequatorial position. In an embodiment, the A recognition elements arelinked to a framework at a pendant position and the B recognitionelements are linked to the framework at an equatorial position.

Although not limiting to the present invention, it is believed that theA and B recognition elements represent the assortment of functionalgroups and geometric configurations employed by polypeptide receptors.Although not limiting to the present invention, it is believed that theA recognition elements represent six advantageous functional groups orconfigurations and that the addition of functional groups to several ofthe aryl groups increases the range of possible binding interactions.Although not limiting to the present invention, it is believed that theB recognition elements represent six advantageous functional groups, butin different configurations than employed for the A recognitionelements. Although not limiting to the present invention, it is furtherbelieved that this increases the range of binding interactions andfurther extends the range of functional groups and configurations thatis explored by molecular configurations of the building blocks.

In an embodiment, the building blocks including the A and B recognitionelements can be visualized as occupying a binding space defined bylipophilicity/hydrophilicity and volume. A volume can be calculated(using known methods) for each building block including the various Aand B recognition elements. A measure of lipophilicity/hydrophilicity(logP) can be calculated (using known methods) for each building blockincluding the various A and B recognition elements. Negative values oflogP show affinity for water over nonpolar organic solvent and indicatea hydrophilic nature. A plot of volume versus logP can then show thedistribution of the building blocks through a binding space defined bysize and lipophilicity/hydrophilicity.

Reagents that form many of the recognition elements are commerciallyavailable. For example, reagents for forming recognition elements A1,A2, A3, A3a, A4, A5, A6, A7, A8, A9 B1, B2, B3, B3a, B4, B5, B6, B7, B8,and B9 are commercially available.

Linkers

The linker is selected to provide a suitable coupling of the buildingblock to a support. The framework can interact with the ligand as partof the artificial receptor. The linker can also provide bulk, distancefrom the support, hydrophobicity, hydrophilicity, and like structuralcharacteristics to the building block. Coupling building blocks to thesupport can employ covalent bonding or noncovalent interactions.Suitable noncovalent interactions include interactions between ions,hydrogen bonding, van der Waals interactions, and the like. In anembodiment, the linker includes moieties that can engage in covalentbonding or noncovalent interactions. In an embodiment, the linkerincludes moieties that can engage in covalent bonding. Suitable groupsfor forming covalent and reversible covalent bonds are describedhereinabove.

Linkers for Reversibly Immobilizable Building Blocks

The linker can be selected to provide suitable reversible immobilizationof the building block on a support or lawn. In an embodiment, the linkerforms a covalent bond with a functional group on the framework. In anembodiment, the linker also includes a functional group that canreversibly interact with the support or lawn, e.g., through reversiblecovalent bonding or noncovalent interactions.

In an embodiment, the linker includes one or more moieties that canengage in reversible covalent bonding. Suitable groups for reversiblecovalent bonding include those described hereinabove. An artificialreceptor can include building blocks reversibly immobilized on the lawnor support through, for example, imine, acetal, ketal, disulfide, ester,or like linkages. Such functional groups can engage in reversiblecovalent bonding. Such a functional group can be referred to as acovalent bonding moiety, e.g., a second covalent bonding moiety.

In an embodiment, the linker can be functionalized with moieties thatcan engage in noncovalent interactions. For example, the linker caninclude functional groups such as an ionic group, a group that canhydrogen bond, or a group that can engage in van der Waals or otherhydrophobic interactions. Such functional groups can include cationicgroups, anionic groups, lipophilic groups, amphiphilic groups, and thelike.

In an embodiment, the present methods and compositions can employ alinker including a charged moiety (e.g., a second charged moiety).Suitable charged moieties include positively charged moieties andnegatively charged moieties. Suitable positively charged moietiesinclude amines, quaternary ammonium moieties, sulfonium, phosphonium,ferrocene, and the like. Suitable negatively charged moieties (e.g., atneutral pH in aqueous compositions) include carboxylates, phenolssubstituted with strongly electron withdrawing groups (e.g.,tetrachlorophenols), phosphates, phosphonates, phosphinates, sulphates,sulphonates, thiocarboxylates, and hydroxamic acids.

In an embodiment, the present methods and compositions can employ alinker including a group that can hydrogen bond, either as donor oracceptor (e.g., a second hydrogen bonding group). For example, thelinker can include one or more carboxyl groups, amine groups, hydroxylgroups, carbonyl groups, or the like. Ionic groups can also participatein hydrogen bonding.

In an embodiment, the present methods and compositions can employ alinker including a lipophilic moiety (e.g., a second lipophilic moiety).Suitable lipophilic moieties include one or more branched or straightchain C₆₋₃₆ alkyl, C₈₋₂₄ alkyl, C₁₂₋₂₄ alkyl, C₁₂₋₁₈ alkyl, or the like;C₆₋₃₆ alkenyl, C₈₋₂₄ alkenyl, C₁₂₋₂₄ alkenyl, C₁₂₋₁₈ alkenyl, or thelike, with, for example, 1 to 4 double bonds; C₆₋₃₆ alkynyl, C₈₋₂₄alkynyl, C₁₂₋₂₄ alkynyl, C₁₂₋₁₈ alkynyl, or the like, with, for example,1 to 4 triple bonds; chains with 1-4 double or triple bonds; chainsincluding aryl or substituted aryl moieties (e.g., phenyl or naphthylmoieties at the end or middle of a chain); polyaromatic hydrocarbonmoieties; cycloalkane or substituted alkane moieties with numbers ofcarbons as described for chains; combinations or mixtures thereof; orthe like. The alkyl, alkenyl, or alkynyl group can include branching;within chain functionality like an ether group; terminal functionalitylike alcohol, amide, carboxylate or the like; or the like. In anembodiment the linker includes or is a lipid, such as a phospholipid. Inan embodiment, the lipophilic moiety includes or is a 12-carbonaliphatic moiety.

In an embodiment, the linker includes a lipophilic moiety (e.g., asecond lipophilic moiety) and a covalent bonding moiety (e.g., a secondcovalent bonding moiety). In an embodiment, the linker includes alipophilic moiety (e.g., a second lipophilic moiety) and a chargedmoiety (e.g., a second charged moiety).

In an embodiment, the linker forms or can be visualized as forming acovalent bond with an alcohol, phenol, thiol, amine, carbonyl, or likegroup on the framework. Between the bond to the framework and the groupparticipating in or formed by the reversible interaction with thesupport or lawn, the linker can include an alkyl, substituted alkyl,cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl, aryl,heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, a glycoside,or like moiety.

For example, suitable linkers can include: the functional groupparticipating in or formed by the bond to the framework, the functionalgroup or groups participating in or formed by the reversible interactionwith the support or lawn, and a linker backbone moiety. The linkerbackbone moiety can include about 4 to about 48 carbon or heteroatoms,about 8 to about 14 carbon or heteroatoms, about 12 to about 24 carbonor heteroatoms, about 16 to about 18 carbon or heteroatoms, about 4 toabout 12 carbon or heteroatoms, about 4 to about 8 carbon orheteroatoms, or the like. The linker backbone can include an alkyl,substituted alkyl, cycloalkyl, heterocyclic, substituted heterocyclic,aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxyoligomer, a glycoside, mixtures thereof, or like moiety.

In an embodiment, the linker includes a lipophilic moiety, thefunctional group participating in or formed by the bond to theframework, and, optionally, one or more moieties for forming areversible covalent bond, a hydrogen bond, or an ionic interaction. Insuch an embodiment, the lipophilic moiety can have about 4 to about 48carbons, about 8 to about 14 carbons, about 12 to about 24 carbons,about 16 to about 18 carbons, or the like. In such an embodiment, thelinker can include about 1 to about 8 reversible bond/interactionmoieties or about 2 to about 4 reversible bond/interaction moieties.Suitable linkers have structures such as (CH₂)₁COOH, with n=12-24,n=17-24, or n=16-18.

Additional Embodiments of Linkers

The linker can be selected to provide a suitable covalent coupling ofthe building block to a support. The framework can interact with theligand as part of the artificial receptor. The linker can also providebulk, distance from the support, hydrophobicity, hydrophilicity, andlike structural characteristics to the building block. In an embodiment,the linker forms a covalent bond with a functional group on theframework. In an embodiment, before attachment to the support the linkeralso includes a functional group that can be activated to react with orthat will react with a functional group on the support. In anembodiment, once attached to the support, the linker forms a covalentbond with the support and with the framework.

In an embodiment, the linker forms or can be visualized as forming acovalent bond with an alcohol, phenol, thiol, amine, carbonyl, or likegroup on the framework. The linker can include a carboxyl, alcohol,phenol, thiol, amine, carbonyl, maleimide, or like group that can reactwith or be activated to react with the support. Between the bond to theframework and the group formed by the attachment to the support, thelinker can include an alkyl, substituted alkyl, cycloalkyl,heterocyclic, substituted heterocyclic, aryl alkyl, aryl, heteroaryl,heteroaryl alkyl, ethoxy or propoxy oligomer, a glycoside, or likemoiety.

The linker can include a good leaving group bonded to, for example, analkyl or aryl group. The leaving group being “good” enough to bedisplaced by the alcohol, phenol, thiol, amine, carbonyl, or like groupon the framework. Such a linker can include a moiety represented by theformula: R—X, in which X is a leaving group such as halogen (e.g., —Cl,—Br or —I), tosylate, mesylate, triflate, and R is alkyl, substitutedalkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, aglycoside, or like moiety.

Suitable linker groups include those of formula: (CH₂)₁COOH, withn=1-16, n=2-8, n=2-6, or n=3. Reagents that form suitable linkers arecommercially available and include any of a variety of reagents withorthogonal functionality.

Embodiments of Building Blocks

In an embodiment, building blocks can be represented by Formula 2:

in which: RE₁ is recognition element 1, RE₂ is recognition element 2,and L is a linker. X is absent, C═O, CH₂, NR, NR₂, NH, NHCONH, SCONH,CH═N, or OCH₂NH. In certain embodiments, X is absent or C═O. Y isabsent, NH, O, CH₂, or NRCO. In certain embodiments, Y is NH or O. In anembodiment, Y is NH. Z₁ and Z₂ can independently be CH₂, O, NH, S, CO,NR, NR₂, NHCONH, SCONH, CH═N, or OCH₂NH. In an embodiment, Z_(i) and/orZ₂ can independently be O. Z₂ is optional. R₂ is H, CH₃, or anothergroup that confers chirality on the building block and has size similarto or smaller than a methyl group. R₃ is CH₂; CH₂-phenyl; CHCH₃;(CH₂)_(n) with n=2-3; or cyclic alkyl with 3-8 carbons, e.g., 5-6carbons, phenyl, naphthyl. In certain embodiments, R₃ is CH₂ orCH₂-phenyl.

RE₁ is B1, B2, B3, B3a, B4, B5, B6, B7, B8, B9, A1, A2, A3, A3a, A4, A5,A6, A7, A8, or A9. In certain embodiments, RE₁ is B1, B2, B3, B3a, B4,B5, B6, B7, B8, or B9. RE₂ is A1, A2, A3, A3a, A4, A5, A6, A7, A8, A9,B1, B2, B3, B3a, B4, B5, B6, B7, B8, or B9. In certain embodiments, RE₂is A1, A2, A3, A3a, A4, A5, A6, A7, A8, or A9. In an embodiment, RE₁ canbe B2, B3a, B4, B5, B6, B7, or B8. In an embodiment, RE₂ can be A2, A3a,A4, A5, A6, A7, or A8.

In an embodiment, L is the functional group participating in or formedby the bond to the framework (such groups are described herein), thefunctional group or groups participating in or formed by the reversibleinteraction with the support or lawn (such groups are described herein),and a linker backbone moiety. In an embodiment, the linker backbonemoiety is about 4 to about 48 carbon or heteroatom alkyl, substitutedalkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxy oligomer, aglycoside, or mixtures thereof; or about 8 to about 14 carbon orheteroatoms, about 12 to about 24 carbon or heteroatoms, about 16 toabout 18 carbon or heteroatoms, about 4 to about 12 carbon orheteroatoms, about 4 to about 8 carbon or heteroatoms.

In an embodiment, the L is the functional group participating in orformed by the bond to the framework (such groups are described herein)and a lipophilic moiety (such groups are described herein) of about 4 toabout 48 carbons, about 8 to about 14 carbons, about 12 to about 24carbons, about 16 to about 18 carbons. In an embodiment, this L alsoincludes about 1 to about 8 reversible bond/interaction moieties (suchgroups are described herein) or about 2 to about 4 reversiblebond/interaction moieties. In an embodiment, L is (CH₂)_(n)COOH, withn=12-24, n=17-24, or n=16-18.

In an embodiment, L is (CH₂)_(n)COOH, with n=1-16, n=2-8, n=4-6, or n=3.

Building blocks including an A and/or a B recognition element, a linker,and an amino acid framework can be made by methods illustrated ingeneral Scheme 1.

Techniques for Using Artificial Receptors

The present invention includes a method of using artificial receptors.The present invention includes a method of screening candidateartificial receptors to find lead artificial receptors that bind aparticular test ligand. Detecting test ligand bound to a candidateartificial receptor can be accomplished using known methods fordetecting binding to arrays on a slide or to coated tubes or wells. Forexample, the method can employ test ligand labeled with a detectablelabel, such as a fluorophore or an enzyme that produces a detectableproduct. Alternatively, the method can employ an antibody (or otherbinding agent) specific for the test ligand and including a detectablelabel. One or more of the spots that are labeled by the test ligand orthat are more or most intensely labeled with the test ligand areselected as lead artificial receptors. The degree of labeling can beevaluated by evaluating the signal strength from the label. The amountof signal can be directly proportional to the amount of label andbinding. FIG. 13 provides a schematic illustration of an embodiment ofthis process.

According to the present method, screening candidate artificialreceptors against a test ligand can yield one or more lead artificialreceptors. One or more lead artificial receptors can be a workingartificial receptor. That is, the one or more lead artificial receptorscan be useful for detecting the ligand of interest as is. The method canthen employ the one or more artificial receptors as a working artificialreceptor for monitoring or detecting the test ligand. Alternatively, theone or more lead artificial receptors can be employed in the method fordeveloping a working artificial receptor. For example, the one or morelead artificial receptors can provide structural or other informationuseful for designing or screening for an improved lead artificialreceptor or a working artificial receptor. Such designing or screeningcan include making and testing additional candidate artificial receptorsincluding combinations of a subset of building blocks, a different setof building blocks, or a different number of building blocks.

The present invention includes a method of screening candidateartificial receptors to find lead artificial receptors that bind aparticular test ligand. The method can include allowing movement of thebuilding blocks that make up the artificial receptors. Movement ofbuilding blocks can include mobilizing the building block to move alongor on the support and/or to leave the support and enter a fluid (e.g.,liquid) phase separate from the support or lawn.

In an embodiment, building blocks can be mobilized to move along or onthe support (translate or shuffle). Such translation can be employed,for example, to allow building blocks already bound to a test ligand torearrange into a lower energy or tighter binding configuration stillbound to the test ligand. Such translation can be employed, for example,to allow the ligand access to building blocks that are on the supportbut not bound to the ligand. These building blocks can translate intoproximity with and bind to a test ligand.

Building blocks can be induced to move along or on the support or to bereversibly immobilized on the support through any of a variety ofmechanisms. For example, inducing mobility of building blocks caninclude altering the conditions of the support or lawn. That is,altering the conditions can reverse the immobilization of the buildingblocks, thus mobilizing them. Reversibly immobilizing the buildingblocks after they have moved can include, for example, returning to theprevious conditions. Suitable alterations of conditions include changingpH, changing temperature, changing polarity or hydrophobicity, changingionic strength, changing nucleophilicity or electrophilicity (e.g. ofsolvent or solute), and the like.

A building block reversibly immobilized by hydrophobic interactions canbe mobilized by increasing the temperature, by exposing the surface,lawn, or building block to a more hydrophobic solvent (e.g., an organicsolvent or a surfactant), or by reducing ionic strength around thebuilding block. In an embodiment, the organic solvent includesacetonitrile, acetic acid, an alcohol, tetrahydrofuran (THF),dimethylformamide (DMF), hydrocarbons such as hexane or octane, acetone,chloroform, methylene chloride, or the like, or mixture thereof. In anembodiment, the surfactant includes a nonionic surfactant, such as anonylphenol ethoxylate, or the like. A building block that is mobile ona support can be reversibly immobilized by hydrophobic interactions, forexample, by decreasing the temperature, exposing the surface, lawn, orbuilding block to a more hydrophilic solvent (e.g., an aqueous solvent)or increased ionic strength.

A building block reversibly immobilized by hydrogen bonding can bemobilized by increasing the ionic strength, concentration of hydrophilicsolvent, or concentration of a competing hydrogen bonder in the environsof the building block. A building block that is mobile on a support canbe reversibly immobilized through an electrostatic interaction bydecreasing ionic strength of the hydrophilic solvent, or the like.

A building block reversibly immobilized by an electrostatic interactioncan be mobilized by increasing the ionic strength in the environs of thebuilding block. Increasing ionic strength can disrupt electrostaticinteractions. A building block that is mobile on a support can bereversibly immobilized through an electrostatic interaction bydecreasing ionic strength.

A building block reversibly immobilized by an imine, acetal, or ketalbond can be mobilized by decreasing the pH or increasing concentrationof a nucleophilic catalyst in the environs of the building block. In anembodiment, the pH is about 1 to about 4. Imines, acetals, and ketalsundergo acid catalyzed hydrolysis. A building block that is mobile on asupport can be reversibly immobilized by a reversible covalentinteraction, such as by forming an imine, acetal, or ketal bond, byincreasing the pH.

In an embodiment, building blocks can be mobilized to leave the supportand enter a fluid (e.g., liquid) phase separate from the support or lawn(exchange). For example, building blocks can be exchanged onto and/oroff of the support. Exchange can be employed, for example, to allowbuilding blocks on a support but not bound to a test ligand to beremoved from the support. Exchange can be employed, for example, to addadditional building blocks to the support. The added building blocks canhave structures selected based on knowledge of the structures of thebuilding blocks in artificial receptors that bind the test ligand. Theadded building blocks can have structures selected to provide additionalstructural diversity. The added building blocks can include all of thebuilding blocks.

A building block reversibly immobilized by hydrophobic interactions canbe released from the support by, for example, raising the temperature,e.g., of the support and/or artificial receptor. For example, thehydrophobic interactions (e.g., the hydrophobic group on the support orlawn and on the building block) can be selected to provide immobilizedbuilding block at about room temperature or below and release can beaccomplished at a temperature above room temperature. For example, thehydrophobic interactions can be selected to provide immobilized buildingblock at about refrigerator temperature (e.g., 4° C.) or below andrelease can be accomplished at a temperature of, for example, roomtemperature or above. By way of further example, a building block can bereversibly immobilized by hydrophobic interactions, for example, bycontacting the surface or artificial receptor with a fluid containingthe building block and that is at or below room temperature.

A building block reversibly immobilized by hydrophobic interactions canbe released from the support by, for example, contacting the artificialreceptor with a sufficiently hydrophobic fluid (e.g., an organic solventor a surfactant). In an embodiment, the organic solvent includesacetonitrile, acetic acid, an alcohol, tetrahydrofuran (THF),dimethylformamide (DMF), hydrocarbons such as hexane or octane, acetone,chloroform, methylene chloride, or the like, or mixture thereof. In anembodiment, the surfactant includes a nonionic surfactant, such as anonylphenol ethoxylate, or the like. Such reversible immobilization canalso be effected by contacting the surface or artificial receptor with ahydrophilic solvent and allowing the somewhat lipophilic building blockto partition on to the hydrophobic surface or lawn.

A building block reversibly immobilized by an imine, acetal, or ketalbond can be released from the support by, for example, contacting theartificial receptor with fluid having an acid pH or including anucleophilic catalyst. In an embodiment, the pH is about 1 to about 4. Abuilding block can be reversibly immobilized by a reversible covalentinteraction, such as by forming an imine, acetal, or ketal bond, bycontacting the surface or artificial receptor with fluid having aneutral or basic pH.

A building block reversibly immobilized by an electrostatic interactioncan be released by, for example, contacting the artificial receptor withfluid having sufficiently high ionic strength to disrupt theelectrostatic interaction. A building block can be reversiblyimmobilized through an electrostatic interaction by contacting thesurface or artificial receptor with fluid having ionic strength thatpromotes electrostatic interaction between the building block and thesupport and/or lawn.

Test Ligands

The test ligand can be any ligand for which binding to an array orsurface can be detected. The test ligand can be a pure compound, amixture, or a “dirty” mixture containing a natural product or pollutant.Such dirty mixtures can be tissue homogenate, biological fluid, soilsample, water sample, or the like.

Test ligands include prostate specific antigen, other cancer markers,insulin, warfarin, other anti-coagulants, cocaine, other drugs-of-abuse,markers for E. coli, markers for Salmonella sp., markers for otherfood-borne toxins, food-borne toxins, markers for Smallpox virus,markers for anthrax, markers for other possible toxic biological agents,pharmaceuticals and medicines, pollutants and chemicals in hazardouswaste, toxic chemical agents, markers of disease, pharmaceuticals,pollutants, biologically important cations (e.g., potassium or calciumion), peptides, carbohydrates, enzymes, bacteria, viruses, mixturesthereof, and the like. In certain embodiments, the test ligand can be atleast one of small organic molecules, inorganic/organic complexes, metalion, mixture of proteins, protein, nucleic acid, mixture of nucleicacids, mixtures thereof, and the like.

Suitable test ligands include any compound or category of compoundsdescribed elsewhere in this document as being a test ligand, including,for example, the microbes, proteins, cancer cells, drugs of abuse, andthe like described above.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1 Synthesis of Building Blocks

Selected building blocks representative of the alkyl-aromatic-polar spanof the an embodiment of the building blocks were synthesized anddemonstrated effectiveness of these building blocks for making candidateartificial receptors. These building blocks were made on a frameworkthat can be represented by tyrosine and included numerous recognitionelement pairs. These recognition element pairs were selected along thediagonal of Table 2, and include enough of the range from alkyl, toaromatic, to polar to represent a significant degree of the interactionsand functional groups of the full set of 81 such building blocks.

Synthesis

Building block synthesis employed a general procedure outlined in Scheme7, which specifically illustrates synthesis of a building block on atyrosine framework with recognition element pair A4B4. This generalprocedure was employed for synthesis of building blocks includingTyrA1B1 [1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA4B2, TyrA4B4,TyrA4B6, TyrA4B8, TyrA6B2, TyrA6B4, TyrA6B6, TyrA6B8, TyrA8B2, TyrA8B4,TyrA8B6, TyrA8B8, and TyrA9B9, respectively.

Results

Synthesis of the desired building blocks proved to be generallystraightforward. These syntheses illustrate the relative simplicity ofpreparing the building blocks with 2 recognition elements havingdifferent structural characteristics or structures (e.g. A4B2, A6B3,etc.) once the building blocks with corresponding recognition elements(e.g. A2B2, A4B4, etc) have been prepared via their X BOC intermediate.

The conversion of one of these building blocks to a building block witha lipophilic linker can be accomplished by reacting the activatedbuilding block with, for example, dodecyl amine.

Example 2 Preparation and Evaluation of Microarrays of CandidateArtificial Receptors

Microarrays of candidate artificial receptors were made and evaluatedfor binding several protein ligands. The results obtained demonstratethe 1) the simplicity with which microarrays of candidate artificialreceptors can be prepared, 2) binding affinity and binding patternreproducibility, 3) significantly improved binding for building blockheterogeneous receptor environments when compared to the respectivehomogeneous controls, and 4) ligand distinctive binding patterns (e.g.,working receptor complexes).

Materials and Methods

Building blocks were synthesized and activated as described inExample 1. The building blocks employed in this example were TyrA1B1[1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B2,TyrA6B4, and TyrA6B6. The abbreviation for the building block includinga linker, a tyrosine framework, and recognition elements AxBy isTyrAxBy.

Microarrays for the evaluation of the 130 n=2 and n=3, and forevaluation of the 273 n=2, n=3, and n=4, candidate receptor environmentswere prepared as follows by modifications of known methods. Briefly:Amine modified (amine “lawn”; SuperAmine Microarray plates) microarrayplates were purchased from Telechem Inc., Sunnyvale, Calif.(www.arrayit.com). These plates were manufactured specifically formicroarray preparation and had a nominal amine load of 2-4 amines persquare nm according to the manufacturer. The CAM microarrays wereprepared using a pin microarray spotter instrument from Telechem Inc.(SpotBot™ Arrayer) typically with 200 um diameter spotting pins fromTelechem Inc. (Stealth Micro Spotting Pins, SMP6) and 400-420 um spotspacing.

The 9 building blocks were activated in aqueous dimethylformamide (DMF)solution as described above. For preparing the 384-well feed plate, theactivated building block solutions were diluted 10-fold with a solutionof DMF/H₂O/PEG400 (90/10/10, v/v/v; PEG400 is polyethylene glycolnominal 400 FW, Aldrich Chemical Co., Milwaukee, Wis.). These stocksolutions were aliquotted (10 μl per aliquot) into the wells of a384-well microwell plate (Telechem Inc.). A separate series of controlswere prepared by aliquotting 10 μl of building block with either 10 μlor 20 μl of the activated [1-1] solution. The plate was covered withaluminum foil and placed on the bed of a rotary shaker for 15 minutes at1,000 RPM. This master plate was stored covered with aluminum foil at−20° C. when not in use.

For preparing the 384-well SpotBot™ plate, a well-to-well transfer (e.g.A-1 to A-1, A-2 to A-2, etc.) from the feed plate to a second 384-wellplate was performed using a 4 μl transfer pipette. This plate was storedtightly covered with aluminum foil at −20° C. when not in use. TheSpotBot™ was used to prepare up to 13 microarray plates per run usingthe 4 μl microwell plate. The SpotBot™ was programmed to spot from eachmicrowell in quadruplicate. The wash station on the SpotBot™ used a washsolution of EtOH/H₂O (20/80, v/v). This wash solution was also used torinse the microarrays on completion of the SpotBot™ printing run. Theplates were given a final rinse with deionized (DI) water, dried using astream of compressed air, and stored at room temperature.

Certain of the microarrays were further modified by reacting theremaining amines with succinic anhydride to form a carboxylate lawn inplace of the amine lawn.

The following test ligands and labels were used in these experiments:

1) r-Phycoerythrin, a commercially available and intrinsicallyfluorescent protein with a FW of 2,000,000.

2) Ovalbumin labeled with the Alexa™ fluorophore (Molecular Probes Inc.,Eugene, Oreg.).

3) BSA, bovine serum albumin, labeled with activated Rhodamine (PierceChemical, Rockford, Ill.) using the known activated carboxyl protocol.BSA has a FW of 68,000; the material used for this study had ca. 1.0rhodamine per BSA.

4) Horseradish peroxidase (HRP) modified with extra amines and labeledas the acetamide derivative or with a 2,3,7,8-tetrachlorodibenzodixoinderivative were available through known methods. Fluorescence detectionof these HRP conjugates was based on the Alexa 647-tyramide kitavailable from Molecular Probes, Eugene, Oreg.

5) Cholera toxin.

Microarray incubation and analysis was conducted as follows: For testligand incubation with the microarrays, solutions (e.g. 500 μl) of thetarget proteins in PBS-T (PBS with 20 μl/L of Tween-20) at typicalconcentrations of 10, 1.0 and 0.1 μg/ml were placed onto the surface ofa microarray and allowed to react for, e.g., 30 minutes. The microarraywas rinsed with PBS-T and DI water and dried using a stream ofcompressed air.

The incubated microarray was scanned using an Axon Model 4200AFluorescence Microarray Scanner (Axon Instruments, Union City, Calif.).The Axon scanner and its associated software produce a false color16-bit image of the fluorescence intensity of the plate. This 16-bitdata is integrated using the Axon software to give a Fluorescence Unitsvalue (range 0-65,536) for each spot on the microarray. This data isthen exported into an Excel file (Microsoft) for further analysisincluding mean, standard deviation and coefficient of variationcalculations.

Results

The CARA™: Combinatorial Artificial Receptor Array™ concept has beendemonstrated using a microarray format. A CARA microarray based on N=9building blocks was prepared and evaluated for binding to severalprotein and substituted protein ligands. This microarray included 144candidate receptors (18 n=1 controls plus 6 blanks; 36 n=2 candidatereceptors; 84 n=3 candidate receptors). This microarray demonstrated: 1)the simplicity of CARA microarray preparation, 2) binding affinity andbinding pattern reproducibility, 3) significantly improved binding forbuilding block heterogeneous receptor environments when compared to therespective homogeneous controls, and 4) ligand distinctive bindingpatterns.

Reading the Arrays

A typical false color/gray scale image of a microarray that wasincubated with 2.0 μg/ml r-phycoerythrin is shown in FIG. 23. This imageillustrates that the processes of both preparing the microarray andprobing it with a protein test ligand produced the expected range ofbinding as seen in the visual range of relative fluorescence from darkto bright spots.

The starting point in analysis of the data was to take the integratedfluorescence units data for the array of spots and normalize to theobserved value for the [1-1] building block control. Subsequent analysisincluded mean, standard deviation and coefficient of variationcalculations. Additionally, control values for homogeneous buildingblocks were obtained from the building block plus [1-1] data.

First Set of Experiments

The following protein ligands were evaluated for binding to thecandidate artificial receptors in the microarray. The resultingFluorescence Units versus candidate receptor environment data ispresented in both a 2D format where the candidate receptors are placedalong the X-axis and the Fluorescence Units are shown on the Y-axis anda 3D format where the Candidate Receptors are placed in an X-Y formatand the Fluorescence Units are shown on the Z-axis. A key for thecomposition of each spot was developed (not shown). A key for thebuilding blocks in each of the 2D and 3D representations of the resultswas also developed (not shown). The data presented are for 1-2 μg/mlprotein concentrations.

FIGS. 24 and 25 illustrate binding data for r-phycoerythrin (intrinsicfluorescence). FIGS. 26 and 27 illustrate binding data for ovalbumin(commercially available with fluorescence label). FIGS. 28 and 29illustrate binding data for bovine serum albumin (labeled withrhodamine). FIGS. 30 and 31 illustrate binding data for HRP-NH-Ac(fluorescent tyramide read-out). FIGS. 32 and 33 illustrate binding datafor HRP-NH-TCDD (fluorescent tyramide read-out).

These results demonstrate not only the application of the CARAmicroarray to candidate artificial receptor evaluation but also a few ofthe many read-out methods (e.g. intrinsic fluorescence, fluorescentlylabeled, in situ fluorescence labeling) which can be utilized for highthroughput candidate receptor evaluation.

The evaluation of candidate receptors benefits from reproducibility. Thefollowing results demonstrate that the present microarrays providedreproducible ligand binding.

The microarrays were printed with each combination of building blocksspotted in quadruplicate. Visual inspection of a direct plot (FIG. 34)of the raw fluorescence data (from the run illustrated in FIG. 23) forone block of binding data obtained for r-phycoerythrin demonstrates thatthe candidate receptor environment “spots” showed reproducible bindingto the test ligand. Further analysis of the r-phycoerythrin data (FIG.23) led to only 9 out of 768 spots (1.2%) being deleted as outliers.Analysis of the r-phycoerythrin quadruplicate data for the entire arraygives a mean standard deviation for each experimental quadruplicate setof 938 fluorescence units, with a mean coefficient of variation of19.8%.

Although these values are acceptable, a more realistic comparisonemployed the standard deviation and coefficient of variation of the morestrongly bound, more fluorescent receptors. The overall mean standarddeviation unrealistically inflates the coefficient of variation for theweakly bound, less fluorescent receptors. The coefficient of variationfor the 19 receptors with greater than 10,000 Fluorescent Units of boundtarget is 11.1%, which is well within the range required to producemeaningful binding data.

One goal of the CARA approach is the facile preparation of a significantnumber of candidate receptors through combinations of structurallysimple building blocks. The following results establish that both theindividual building blocks and combinations of building blocks have asignificant, positive effect on test ligand binding.

The binding data illustrated in FIGS. 23-33 demonstrate thatheterogeneous combinations of building blocks (n=2, n=3) aredramatically superior candidate receptors made from a single buildingblock (n=1). For example, FIG. 25 illustrate both the diversity ofbinding observed for n=2, n=3 candidate receptors with fluorescent unitsranging from 0 to ca. 40,000. These data also illustrate and the ca.10-fold improvement in binding affinity obtained upon going from thehomogeneous (n=1) to heterogeneous (n=2, n=3) receptor environments.

The effect of heterogeneous building blocks is most easily observed bycomparing selected n=3 receptor environments candidate receptorsincluding 1 or 2 of those building blocks (their n=2 and n=1 subsets).FIGS. 35 and 36 illustrate this comparison for two different n=3receptor environments using the r-phycoerythrin data. In these examples,it is clear that progression from the homogeneous system (n=1) to theheterogeneous systems (n=2, n=3) produces significantly enhancedbinding.

Although van der Waals interactions are an important part of molecularrecognition, it is important to establish that the observed binding isnot a simple case of hydrophobic/hydrophilic partitioning. That is, thatthe observed binding was the result of specific interactions between theindividual building blocks and the target The simplest way to evaluatethe effects of hydrophobicity and hydrophilicity is to compare buildingblock logP value with observed binding. LogP is a known and acceptedmeasure of lipophilicity, which can be measured or calculated by knownmethods for each of the building blocks. FIGS. 37 and 38 establish thatthe observed target binding, as measured by fluorescence units, is notdirectly proportional to building block logP. The plots in FIGS. 37 and38 illustrate a non-linear relationship between binding (fluorescenceunits) and building block logP.

One advantage of the present methods and arrays is that the ability toscreen large numbers of candidate receptor environments will lead to acombination of useful target affinities and to significant targetbinding diversity. High target affinity is useful for specific targetbinding, isolation, etc. while binding diversity can provide multiplexedtarget detection systems. This example employed a relatively smallnumber of building blocks to produce ca. 120 binding environments. Thefollowing analysis of the present data clearly demonstrates that even arelatively small number of binding environments can produce diverse anduseful artificial receptors.

The target binding experiments performed for this study used proteinconcentrations including 0.1 to 10 μg/ml. Considering the BSA data asrepresentative, it is clear that some of the receptor environmentsreadily bound 1.0 ug/ml BSA concentrations near the saturation valuesfor fluorescence units (see, e.g., FIG. 29). Based on these data and theformula weight of 68,000 for BSA, several of the receptor environmentsreadily bind BSA at ca. 15 picomole/ml or 15 nanomolar concentrations.Additional experiments using lower concentrations of protein (data notshown) indicate that, even with a small selection of candidate receptorenvironments, femptomole/ml or picomolar detection limits have beenattained.

One goal of artificial receptor development is the specific recognitionof a particular target. FIG. 39 compares the observed binding forr-phycoerythrin and BSA. Comparison of the overall binding patternindicates some general similarities. However, comparison of specificfeatures of binding for each receptor environment demonstrates that thetwo targets have distinctive recognition features as indicated by the(*) in FIG. 39.

One goal of artificial receptor development is to develop receptorswhich can be used for the multiplexed detection of specific targets.Comparison of the r-phycoerythrin, BSA and ovalbumin data from thisstudy (FIGS. 25, 27, 29) were used to select representative artificialreceptors for each target. FIGS. 40, 41 and 42 employ data obtained inthe present example to illustrate identification of each of these threetargets by their distinctive binding patterns.

Conclusions

The optimum receptor for a particular target requires molecularrecognition which is greater than the expected sum of the individualhydrophilic, hydrophobic, ionic, etc. interactions. Thus, theidentification of an optimum (specific, sensitive) artificial receptorfrom the limited pool of candidate receptors explored in this prototypestudy, was not expected and not likely. Rather, the goal was todemonstrate that all of the key components of the CARA: CombinatorialArtificial Receptor Array concept could be assembled to form afunctional receptor microarray. This goal has been successfullydemonstrated.

This study has conclusively established that CARA microarrays can bereadily prepared and that target binding to the candidate receptorenvironments can be used to identify artificial receptors and testligands. In addition, these results demonstrate that there issignificant binding enhancement for the building block heterogeneous(n=2, n=3, or n=4) candidate receptors when compared to theirhomogeneous (n=1) counterparts. When combined with the binding patternrecognition results and the demonstrated importance of both theheterogeneous receptor elements and heterogeneous building blocks, theseresults clearly demonstrate the significance of the CARA CandidateArtificial Receptor−>Lead Artificial Receptor−>Working ArtificialReceptor strategy.

Example 3 Preparation and Evaluation of Microarrays of CandidateArtificial Receptors Including Reversibly Immobilized Building Blocks

Microarrays of candidate artificial receptors including building blocksimmobilized through van der Waals interactions were made and evaluatedfor binding of a protein ligand. The evaluation was conducted at severaltemperatures, above and below a phase transition temperature for thelawn (vide infra).

Materials and Methods

Building blocks 2-2, 2-4, 2-6, 4-2, 4-4, 4-6, 6-2, 6-4, 6-6 whereprepared as described in Example 1. The C12 amide was prepared using thepreviously described carbodiimide activation of the carboxyl followed byaddition of dodecylamine. This produced a building block with a 12carbon alkyl chain linker for reversible immobilization in the C18 lawn.

Amino lawn microarray plates (Telechem) were modified to produce the C18lawn by reaction of stearoyl chloride (Aldrich Chemical Co.) in A)dimethylformamide/PEG 400 solution (90:10, v/v, PEG 400 is polyethyleneglycol average MW 400 (Aldrich Chemical Co.) or B) methylenechloride/TEA solution (100 ml methylene chloride, 200 μl triethylamine)using the lawn modification procedures generally described in Example 2.

The C18 lawn plates where printed using the SpotBot standard procedureas described in Example 2. The building blocks were in printingsolutions prepared by solution of ca. 10 mg of each building block in300 μl of methylene chloride and 100 μl methanol. To this stock wasadded 900 μl of dimethylformamide and 100 μl of PEG 400. The 36combinations of the 9 building blocks taken two at a time (N9:n2, 36combinations) where prepared in a 384-well microwell plate which wasthen used in the SpotBot to print the microarray in quadruplicate. Arandom selection of the print positions contained only print solution.

The selected microarray was incubated with a 1.0 μg/ml solution of thetest ligand, cholera toxin subunit B labeled with the Alexa™ fluorophore(Molecular Probes Inc., Eugene, Oreg.), using the followingvariables: 1) the microarray was washed with methylene chloride, ethanoland water to create a control plate; and 2) the microarray was incubatedat 4° C., 23° C., or 44° C. After incubation, the plate(s) were rinsedwith water, dried and scanned (AXON 4100A). Data analysis was asdescribed in Example 2.

Results

A control array from which the building blocks had been removed bywashing with organic solvent did not bind cholera toxin (FIG. 43). FIGS.44-46 illustrate fluorescence signals from arrays printed identically,but incubated with cholera toxin at 4° C., 23° C., or 44° C.,respectively. Spots of fluorescence can be seen in each array, with verypronounced spots produced by incubation at 44° C. The fluorescencevalues for the spots in each of these three arrays are shown in FIGS.47-49. Fluorescence signal generally increases with temperature, withmany nearly equally large signals observed after incubation at 44° C.Linear increases with temperature can reflect expected improvements inbinding with temperature. Nonlinear increases reflect rearrangement ofthe building blocks on the surface to achieve improved binding, whichoccurred above the phase transition for the lipid surface (vide infra).

FIG. 50 can be compared to FIG. 48. The fluorescence signals plotted inFIG. 48 resulted from binding to reversibly immobilized building blockson a support at 23° C. The fluorescence signals plotted in FIG. 50resulted from binding to covalently immobilized building blocks on asupport at 23° C. These figures compare the same combinations ofbuilding blocks in the same relative positions, but immobilized in twodifferent ways.

The binding to covalently immobilized building blocks was also evaluatedat 4° C., 23° C., or 44° C. FIG. 51 illustrates the changes influorescence signal from individual combinations of covalentlyimmobilized building blocks at 4° C., 23° C., or 44° C. Bindingincreased modestly with temperature. The mean increase in binding was1.3-fold. A plot of the fluorescence signal for each of the covalentlyimmobilized artificial receptors at 23° C. against its signal at 44° C.(not shown) yields a linear correlation with a correlation coefficientof 0.75. This linear correlation indicates that the mean 1.3-foldincrease in binding is a thermodynamic effect and not optimization ofbinding.

FIG. 52 illustrates the changes in fluorescence signal from individualcombinations of reversibly immobilized building blocks at 4° C., 23° C.,or 44° C. This graph illustrates that at least one combination ofbuilding blocks (candidate artificial receptor) exhibited a signal thatremained constant as temperature increased. At least one candidateartificial receptor exhibited an approximately linear increase in signalas temperature increased. Such a linear increase indicates normaltemperature effects on binding. The candidate artificial receptor withthe lowest binding signal at 4° C. became one of the best binders at 44°C. This indicates that rearrangement of the building blocks of thisreceptor above the phase transition for the lawn, which increases thebuilding blocks' mobility, produced increased binding. Other receptorscharacterized by greater changes in binding between 23° C. and 44° C.(compared to between 4° C. and 23° C.) also underwent dynamic affinityoptimization.

FIG. 53 illustrates the data presented in FIG. 51 (lines marked A) andthe data presented in FIG. 52 (lines marked B). The increases in bindingobserved with the reversibly immobilized building blocks aresignificantly greater than the increases observed with covalently boundbuilding blocks. Binding to reversibly immobilized building blocksincreased from 23° C. and 44° C. by a median value of 6.1-fold and amean value of 24-fold. This confirms that movement of the reversiblyimmobilized building blocks within the receptors increased binding(i.e., the receptor underwent dynamic affinity optimization).

A plot of the fluorescence signal for each of the reversibly immobilizedartificial receptors at 23° C. against its signal at 44° C. (not shown)yields no correlation (correlation coefficient of 0.004). A plot of thefluorescence signal for each of the reversibly immobilized artificialreceptors at 44° C. against the signal for the corresponding covalentlyimmobilized receptor (not shown) also yields no correlation (correlationcoefficient 0.004). This lack of correlation provides further evidencethat movement of the reversibly immobilized building blocks within thereceptors increased binding.

FIG. 54 illustrates a graph of the fluorescence signal at 44° C. dividedby the signal at 23° C. against the fluorescence signal obtained frombinding at 23° C. for the artificial receptors with reversiblyimmobilized receptors. This comparison indicates that the bindingenhancement is independent of the initial affinity of the receptor forthe test ligand.

Table 1 identifies the reversibly immobilized building blocks making upeach of the artificial receptors, lists the fluorescence signal (bindingstrength) at 44° C. and 23° C., and the ratios of the observed bindingat these two temperatures. These data illustrate that each artificialreceptor reflects a unique attribute for each combination of buildingblocks relative to the role of each individual building block.

TABLE 1 Building Blocks Making Up Signal at Ratio of Signals, Receptor44° C. Signal at 23° C. 44° C./23° C. 22 24 24136 4611 5.23 22 26 1666043 387.44 22 42 17287 −167 −103.51 22 44 16726 275 60.82 22 46 250163903 6.41 22 62 13990 3068 4.56 22 64 15294 3062 4.99 22 66 11980 36273.30 24 26 22688 1291 17.57 24 42 26808 −662 −40.50 24 44 23154 90425.61 24 46 42197 2814 15.00 24 62 19374 2567 7.55 24 64 27599 262105.34 24 66 16238 5334 3.04 26 42 22282 4974 4.48 26 44 26240 530 49.5126 46 23144 4273 5.42 26 62 29022 4920 5.90 26 64 23416 5551 4.22 26 6619553 5353 3.65 42 44 29093 6555 4.44 42 46 18637 3039 6.13 42 62 226434853 4.67 42 64 20836 6343 3.28 42 66 14391 9220 1.56 44 46 25600 32667.84 44 62 15544 4771 3.26 44 64 25842 3073 8.41 44 66 22471 5142 4.3746 62 32764 8522 3.84 46 64 21901 3343 6.55 46 66 23516 3742 6.28 62 6424069 7149 3.37 62 66 15831 2424 6.53 64 66 21310 2746 7.76

Conclusions

This experiment demonstrated that an array including reversiblyimmobilized building blocks binds a protein substrate, like an arraywith covalently immobilized building blocks. The binding increasednonlinearly as temperature increased, indicating that movement of thebuilding blocks increased binding. Many of the candidate artificialreceptors demonstrated improved binding upon mobilization of thebuilding blocks.

Example 4 The Oligosaccharide Portion of GM1 Competes with ArtificialReceptors for Binding to Cholera Toxin

Microarrays of candidate artificial receptors were made and evaluatedfor binding of cholera toxin. The arrays were also evaluated fordisrupting that binding. Disrupting of binding employed a compound thatbinds to cholera toxin, the oligosaccharide moiety from GM1 (GM1 OS).The results obtained demonstrate that a ligand of a protein specificallydisrupted binding of the protein to the microarray.

Materials and Methods

Building blocks were synthesized and activated as described inExample 1. The building blocks employed in this example were TyrA1B1[1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA2B8, TyrA3B3, TyrA3B5, TyrA3B7,TyrA4B2, TyrA4B4, TyrA4B6, TyrA4B8, TyrA5B3, TyrA5B5, TyrA5B7, TyrA6B2,TyrA6B4, TyrA6B6, TyrA6B8, TyrA7B3, TyrA7B5, TyrA7B7, TyrA8B2, TyrA8B4,TyrA8B6, and TyrA8B8. The abbreviation for the building block includinga linker, a tyrosine framework, and recognition elements AxBy isTyrAxBy.

Microarrays for the evaluation of the 171 n=2 candidate receptorenvironments were prepared as follows by modifications of known methods.An “n=2” receptor environment includes two different building blocks.Briefly: Amine modified (amine “lawn”; SuperAmine Microarray plates)microarray plates were purchased from Telechem Inc., Sunnyvale, Calif.These plates were manufactured specifically for microarray preparationand had a nominal amine load of 2-4 amines per square nm according tothe manufacturer. The microarrays were prepared using a pin microarrayspotter instrument from Telechem Inc. (SpotBot™ Arrayer) typically with200 μm diameter spotting pins from Telechem Inc. (Stealth Micro SpottingPins, SMP6) and 400-420 μm spot spacing.

The 19 building blocks were activated in aqueous dimethylformamide (DMF)solution as described above. For preparing the 384-well feed plate, theactivated building block solutions were diluted 10-fold with a solutionof DMF/H₂O/PEG400 (90/10/10, v/v/v; PEG400 is polyethylene glycolnominal 400 FW, Aldrich Chemical Co., Milwaukee, Wis.). These stocksolutions were aliquotted (10 μl per aliquot) into the wells of a384-well microwell plate (Telechem Inc.). Control spots included thebuilding block [1-1]. The plate was covered with aluminum foil andplaced on the bed of a rotary shaker for 15 minutes at 1,000 RPM. Thismaster plate was stored covered with aluminum foil at −20° C. when notin use.

For preparing the 384-well SpotBot™ plate, a well-to-well transfer (e.g.A-1 to A-1, A-2 to A-2, etc.) from the feed plate to a second 384-wellplate was performed using a 4 μl transfer pipette. This plate was storedtightly covered with aluminum foil at −20° C. when not in use. TheSpotBot™ was used to prepare up to 13 microarray plates per run usingthe 4 μl microwell plate. The SpotBot™ was programmed to spot from eachmicrowell in quadruplicate. The wash station on the SpotBot™ used a washsolution of EtOH/H₂O (20/80, v/v). This wash solution was adjusted to pH4 with 1 M HCl and used to rinse the microarrays on completion of theSpotBot™ printing run. The plates were given a final rinse withdeionized (DI) water, dried using a stream of compressed air, and storedat room temperature. The microarrays were further modified by reactingthe remaining amines with acetic anhydride to form an acetamide lawn inplace of the amine lawn.

The test ligand employed in these experiments was cholera toxin labeledwith the Alexa™ fluorophore (Molecular Probes Inc., Eugene, Oreg.). Thecandidate disruptor employed in these experiments was GM1 OS (GM1oligosaccharide), a known ligand for cholera toxin.

Microarray incubation and analysis was conducted as follows: For controlincubations with the microarrays, solutions (e.g. 500 μl) of the choleratoxin in PBS-T (PBS with 20 μl/L of Tween-20) at a concentrations of 1.7pmol/ml (0.1 μg/ml) was placed onto the surface of a microarray andallowed to react for 30 minutes. For disruptor incubations with themicroarrays, solutions (e.g. 500 μl) of the cholera toxin (1.7 pmol/ml,0.1 μg/ml) and the desired concentration of GM1 OS in PBS-T (PBS with 20μl/L of Tween-20) was placed onto the surface of a microarray andallowed to react for 30 minutes. GM1 OS was added at 0.34 and at 5.1 μMin separate experiments. After either of these incubations, themicroarray was rinsed with PBS-T and DI water and dried using a streamof compressed air.

The incubated microarray was scanned using an Axon Model 4200AFluorescence Microarray Scanner (Axon Instruments, Union City, Calif.).The Axon scanner and its associated software produce a false color16-bit image of the fluorescence intensity of the plate. This 16-bitdata is integrated using the Axon software to give a Fluorescence Unitsvalue (range 0-65,536) for each spot on the microarray. This data isthen exported into an Excel file (Microsoft) for further analysisincluding mean, standard deviation and coefficient of variationcalculations.

Table 2 identifies the building blocks in each of the first 150 receptorenvironments.

TABLE 2 Building Blocks 1 22 24 2 22 28 3 22 42 4 22 46 5 22 55 6 22 647 22 68 8 22 82 9 22 86 10 24 26 11 24 33 12 24 44 13 26 77 14 26 84 1526 88 16 28 42 17 22 26 18 22 33 19 22 44 20 22 48 21 22 62 22 22 66 2322 77 24 22 84 25 22 88 26 24 28 27 24 42 28 26 82 29 26 85 30 28 33 3128 44 32 28 46 33 28 55 34 28 64 35 28 68 36 28 82 37 28 86 38 33 42 3933 46 40 42 88 41 44 48 42 44 62 43 44 66 44 44 77 45 44 84 46 44 88 4746 55 48 28 48 49 28 62 50 28 66 51 28 77 52 28 84 53 28 88 54 33 44 5544 46 56 44 55 57 44 64 58 44 68 59 44 82 60 44 86 61 46 48 62 46 62 6324 46 64 24 55 65 24 64 66 24 68 67 24 82 68 24 86 69 26 28 70 26 42 7126 46 72 26 55 73 26 64 74 26 68 75 33 48 76 33 63 77 33 66 78 33 77 7924 48 80 24 62 81 24 66 82 24 77 83 24 84 84 24 88 85 26 33 86 26 44 8726 48 88 26 62 89 26 66 90 33 55 91 33 64 92 33 68 93 33 82 94 33 84 9533 88 96 42 46 97 42 55 98 42 64 99 42 68 100 42 82 101 42 86 102 46 88103 48 62 104 48 66 105 46 77 106 48 84 107 48 88 108 55 64 109 55 68110 33 86 111 42 44 112 42 48 113 42 62 114 42 66 115 42 77 116 42 84117 48 55 118 48 64 119 48 68 120 48 82 121 48 86 122 55 62 123 55 66124 55 77 125 46 64 126 46 68 127 46 82 128 46 86 129 62 77 130 62 84131 62 88 132 64 68 133 64 82 134 64 86 135 66 68 136 66 82 137 66 86138 68 77 139 68 84 140 68 88 141 46 66 142 46 77 143 46 84 144 62 82145 62 86 146 64 66 147 64 77 148 64 84 149 64 88 150 66 77

Results Low Concentration of GM1 OS

FIG. 55 illustrates binding of cholera toxin to the microarray ofcandidate artificial receptors followed by washing with buffer producedfluorescence signals. These fluorescence signals demonstrate that thecholera toxin bound strongly to certain receptor environments, weakly toothers, and undetectably to some. Comparison to experiments includingthose reported in Example 2 indicates that cholera toxin binding wasreproducible from array to array and from month to month.

Binding of cholera toxin was also conducted with competition from GM1 OS(0.34 μM). FIG. 56 illustrates the fluorescence signals due to choleratoxin binding that were detected after this competition. Notably, manyof the signals illustrated in FIG. 56 are significantly smaller than thecorresponding signals recorded in FIG. 55. The small signals observed inFIG. 56 represent less cholera toxin bound to the array. GM1 OSsignificantly disrupted binding of cholera toxin to many of the receptorenvironments.

The disruption in cholera toxin binding caused by GM1 OS can bevisualized as the ratio of the amount bound in the absence of GM1 OS tothe amount bound in competition with GM1 OS. This ratio is illustratedin FIG. 57. The larger the ratio, the less cholera toxin remained boundto the artificial receptor after competition with GM1 OS. The ratio canbe as large as about 30. The ratios are independent of the quantitybound in the control.

High Concentration of GM1 OS

Binding of cholera toxin to the microarray of candidate artificialreceptors followed by washing with buffer produced fluorescence signalsillustrated in FIG. 58. As before, cholera toxin was reproducible and itbound strongly to certain receptor environments, weakly to others, andundetectably to some. FIG. 59 illustrates the fluorescence signalsdetected due to cholera toxin binding that were detected uponcompetition with GM1 OS at 5.1 μM. Again, GM1 OS significantly disruptedbinding of cholera toxin to many of the receptor environments.

This disruption is presented as the ratio of the amount bound in theabsence of GM1 OS to the amount bound after contacting with GM1 OS inFIG. 60. The ratios range up to about 18 and are independent of thequantity bound in the control.

Conclusions

This experiment demonstrated that binding of a test ligand to anartificial receptor of the present invention can be diminished (e.g.,competed) by a candidate disruptor molecule. In this case the testligand was the protein cholera toxin and the candidate disruptor was acompound known to bind to cholera toxin, GM1 OS. The degree to whichbinding of the test ligand was disrupted was independent of the degreeto which the test ligand bound to the artificial receptor.

Example 5 GM1 Competes With Artificial Receptors for Binding to CholeraToxin

Microarrays of candidate artificial receptors were made and evaluatedfor binding of cholera toxin. The arrays were also evaluated fordisrupting that binding. Disrupting of binding employed a compound thatbinds to cholera toxin, the liposaccharide GM1. The results obtaineddemonstrate that a ligand of a protein specifically disrupts binding ofthe protein to the microarray.

Materials and Methods

Building blocks were synthesized and activated as described inExample 1. The building blocks employed in this example were TyrA1B1[1-1], TyrA2B2, TyrA2B4, TyrA2B6, TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B2,TyrA6B4, and TyrA6B6 in groups of 4 building blocks per artificialreceptor. The abbreviation for the building block including a linker, atyrosine framework, and recognition elements AxBy is TyrAxBy.

Microarrays for the evaluation of the 126 n=4 candidate receptorenvironments were prepared as described above for Example 4. The testligand employed in these experiments was cholera toxin labeled with theAlexa™ fluorophore (Molecular Probes Inc., Eugene, Oreg.). Cholera toxinwas employed at 5.3 nM in both the control and the competitionexperiments. The candidate disruptor employed in these experiments wasGM1, a known ligand for cholera toxin, which competed at concentrationsof 0.042, 0.42, and 8.4 μM. Microarray incubation and analysis wasconducted as described for Example 4.

Table 3 identifies the building blocks in each receptor environment.

TABLE 3 Building Blocks 1 22 24 26 42 2 22 24 26 44 3 22 24 26 46 4 2224 26 61 5 22 24 26 64 6 22 24 26 66 7 22 24 42 44 8 22 24 42 46 9 22 2442 62 10 22 24 42 46 11 22 24 42 66 12 22 24 44 46 13 22 24 44 62 14 2224 44 64 15 22 24 44 66 16 22 24 46 62 17 22 24 46 64 18 22 24 46 66 1922 24 62 64 20 22 24 62 66 21 22 24 64 66 22 22 26 42 44 23 22 26 42 4624 22 26 42 62 25 22 26 42 64 26 22 26 42 66 27 22 26 44 46 28 22 26 4462 29 22 26 44 64 30 22 26 44 66 31 22 26 46 62 32 22 26 46 64 33 22 2646 66 34 22 26 62 64 35 22 26 62 66 36 22 26 64 66 37 22 42 44 46 38 2242 44 62 39 22 42 44 64 40 22 42 44 66 41 22 42 46 62 42 22 42 46 64 4322 42 46 66 44 22 42 62 64 45 22 42 62 66 46 22 42 64 66 47 22 44 46 6248 22 44 46 64 49 22 44 46 66 50 22 44 62 64 51 22 44 62 66 52 22 44 6466 53 22 46 62 64 54 22 46 62 66 55 22 46 64 66 56 22 62 64 66 57 24 2642 44 58 24 26 42 46 59 24 26 42 62 60 24 26 42 64 61 24 26 42 66 62 2426 44 46 63 24 26 44 62 64 24 26 44 64 65 24 26 44 66 66 24 26 46 62 6724 26 46 64 68 24 26 46 66 69 24 26 62 64 70 24 26 62 66 71 24 26 64 6672 24 42 44 46 73 24 42 44 62 74 24 42 44 64 75 24 42 44 66 76 24 42 4662 77 24 42 46 64 78 24 42 46 66 79 24 42 62 64 80 24 42 62 66 81 24 4264 66 82 24 44 46 62 83 24 44 46 64 84 24 44 46 66 85 24 44 62 64 86 2444 62 66 87 24 44 64 66 88 24 46 62 64 89 24 46 62 66 90 24 46 64 66 9124 62 64 66 92 26 42 44 46 93 26 42 44 62 94 26 42 44 64 95 26 42 44 6696 26 42 46 62 97 26 42 46 64 98 26 42 46 66 99 26 42 62 64 100 26 42 6266 101 26 42 64 66 102 26 44 46 62 103 26 44 46 64 104 26 44 46 66 10526 44 62 64 106 26 44 62 66 107 26 44 64 66 108 26 46 62 64 109 26 46 6266 110 26 46 64 66 111 26 62 64 66 112 42 44 46 62 113 42 44 46 64 11442 44 46 66 115 42 44 62 64 116 42 44 62 66 117 42 44 64 66 118 42 46 6264 119 42 46 62 66 120 42 46 64 66 121 42 62 64 66 122 44 46 62 64 12344 46 62 66 124 44 46 64 66 125 44 62 64 66 126 46 62 64 66

Results

FIG. 61 illustrates the fluorescence signals produced by binding ofcholera toxin to the microarray of candidate artificial receptors aloneand in competition with each of the three concentrations of GM1. Themagnitude of the fluorescence signal decreases steadily with increasingconcentration of GM1. The amount of decrease is not quantitativelyidentical for all of the receptors, but each receptor experienceddecreased binding of cholera toxin. These decreases indicate that GM1competed with the artificial receptor for binding to the cholera toxin.

The decreases show a pattern of relative competition for the bindingsite on cholera toxin. This can be demonstrated through graphs offluorescence signal obtained at a particular concentration of GM1against fluorescence signal in the absence of GM1 (not shown). Certainof the receptors appear at similar relative positions on these plots asconcentration of GM1 increases.

The disruption in cholera toxin binding caused by GM1 can be visualizedas the ratio of the amount bound in the absence of GM1 OS to the amountbound upon competition with GM1. This ratio is illustrated in FIG. 62.The larger the ratio, the more cholera toxin remained bound to theartificial receptor upon competition with GM1. The ratio can be as largeas about 14. The ratios are independent of the quantity bound in thecontrol.

Interestingly, in several instances minor changes in structure to theartificial receptor caused significant changes in the ratio. Forexample, the artificial receptor including building blocks 24, 26, 46,and 66 differs from that including 24, 42, 46, and 66 by onlysubstitution of a single building block. (xy indicates building blockTyrAxBy.) The substitution of building block 42 for 26 increased bindingin the presence of GM1 by about 14-fold.

By way of further example, the artificial receptor including buildingblocks 22, 24, 46, and 64 differs from that including 22, 46, 62, and 64by only substitution of a single building block. The substitution ofbuilding block 24 for 62 increased binding in the presence of GM1 byabout 3-fold.

Even substitution of a single recognition element affected binding. Theartificial receptor including building blocks 22, 24, 42, and 44 differsfrom that including 22, 24, 42, and 46 by only substitution of a singlerecognition element. The substitution of building block 44 for 46 (achange of recognition element B6 to B4) increased binding in thepresence of GM1 by about 3-fold.

Conclusions

This experiment demonstrated that binding of a test ligand to anartificial receptor of the present invention can be diminished (e.g.,competed) by a candidate disruptor molecule. In this case the testligand was the protein cholera toxin and the candidate disruptor was acompound known to bind to cholera toxin, GM1. Minor changes in structureof the building blocks making up the artificial receptor causedsignificant changes in the competition.

Example 6 GM1 Employed as a Building Block Alters Binding of CholeraToxin to the Present Artificial Receptors

Microarrays of candidate artificial receptors were made, GM1 was boundto the arrays, and they were evaluated for binding of cholera toxin. Theresults obtained demonstrate that adding GM1 as a building block in anarray of artificial receptors can increase binding to certain of thereceptors.

Materials and Methods

Building blocks were synthesized and activated as described inExample 1. The building blocks employed in this example were thosedescribed in Example 4. Microarrays for the evaluation of the 171 n=2candidate receptor environments were prepared as described above forExample 4. The test ligand employed in these experiments was choleratoxin labeled with the Alexa™ fluorophore (Molecular Probes Inc.,Eugene, Oreg.). Cholera toxin was employed at 0.01 ug/ml (0.17 pM) or0.1 ug/ml (1.7 pM) in both the control and the competition experiments.GM1 was employed as a test ligand for the artificial receptors andbecame a building block for receptors used to bind cholera toxin. Thearrays were contacted with GM1 at either 100 μg/ml, 10 μg/ml, or 1 μg/mlas described above for cholera toxin and then rinsed with deionizedwater. The arrays were then contacted with cholera toxin under theconditions described above. Microarray analysis was conducted asdescribed for Example 4. Table 2 identifies the building blocks in eachreceptor environment.

Results

FIG. 63 illustrates the fluorescence signals produced by binding ofcholera toxin to the microarray of candidate artificial receptorswithout pretreatment with GM1. Binding of GM1 to the microarray ofcandidate artificial receptors followed by binding of cholera toxinproduced fluorescence signals illustrated in FIGS. 64, 65, and 66 (100μg/ml, 10 μg/ml, and 1 μg/ml GM1, respectively).

The enhancement of cholera toxin binding caused by pretreatment with GM1can be visualized as the ratio of the amount bound in the presence ofGM1 to the amount bound in the absence of GM1. This ratio is illustratedin FIG. 67 for 1 μg/ml GM1. The larger the ratio, the more cholera toxinbound to the artificial receptor after pretreatment with GM1. The ratiocan be as large as about 16.

In several instances minor changes in structure to the artificialreceptor caused significant changes in the ratio. For example, theartificial receptor including building blocks 46 and 48 differs fromthat including 46 and 88 by only substitution of a single recognitionelement on a single building block. (xy indicates building blockTyrAxBy.) The substitution of building block 48 for 88 (a change ofrecognition element A8 to A4) increased the ratio representing increasedbinding the presence of GM1 building block from about 0.5 to about 16.Similarly, the artificial receptor including building blocks 42 and 77differs from that including 24 and 77 by only substitution of a singlebuilding block. The substitution of building block 42 for 24 increasedthe ratio representing increased binding the presence of GM1 buildingblock from about 2 to about 14.

Interestingly, several building blocks that exhibited high levels ofbinding of cholera toxin (signals of 45,000 to 65,000 fluorescenceunits) and that include the building block 33 were not strongly affectedby the presence of GM1 as a building block.

Conclusions

This experiment demonstrated that binding of GM1 to an artificialreceptor of the present invention can significantly increase binding bycholera toxin. Minor changes in structure of the building blocks makingup the artificial receptor caused significant changes in the degree towhich GM1 enhanced binding of cholera toxin.

Discussion of Examples 4-6

We have previously demonstrated that an array of working artificialreceptors bind to a protein target in a manner which is complementary tothe specific environment presented by each region of the proteinssurface topology. Thus the pattern of binding of a protein target to anarray of working artificial receptors describes the proteins surfacetopology; including surface structures which participate in e.g.,protein˜small molecule, protein˜peptide, protein-protein,protein˜carbohydrate, protein˜DNA, etc. interactions. It is thuspossible to use the binding of a selected protein to a workingartificial receptor array to characterize these protein˜small molecule,protein˜peptide, protein-protein, protein˜carbohydrate, protein˜DNA,etc. interactions. Moreover, it is possible to utilize the protein toarray interactions to define “leads” for the disruption of theseinteractions.

Cholera Toxin B sub-unit binds to GM1 on the cell surface (structure ofGM1). Studies to identify competitors to this binding event have shownthat competitors to the cholera toxin: GM1 binding interaction (bindingsite) can utilize both a sugar and an alkyl/aromatic functionality(Pickens, et al., Chemistry and Biology, vol. 9, pp 215-224 (2002)). Wehave previously demonstrated that fluorescently labeled Cholera Toxin Bsub-unit binds to arrays of working artificial receptors to give adefined binding pattern which (vida infra) reflects cholera toxin B'ssurface topology. For this study, we sought to demonstrate that thebinding of the cholera toxin to at least some members of the array couldbe disrupted using cholera toxins natural ligand, GM1.

The results presented in the figures clearly demonstrate that thesegoals have been achieved. Specifically, competition between the GM1 OSpentasaccharide or GM1 and a working artificial receptor array forcholera binding clearly gave a binding pattern which was distinct fromthe cholera binding pattern control. Moreover, these resultsdemonstrated the complementarity between several of the workingartificial receptors which contained a naphthyl moiety when compared toworking artificial receptors which only contained phenyl functionality.These results are in keeping with the active site competition studies inPickens, et al. and indicate that the naphthyl and phenyl derivativesrepresent good mimics/probes for the cholera to GM1 interaction. Thespecificity of these interactions was particularly demonstrated by theobservation that the change of a single building block out of 4 in acombination of 4 building blocks system changed a non-competitive to asignificantly competitive environment. These results also indicated thatselected working artificial receptors can be used to develop ahigh-throughput screen for the further evaluation of the cholera: GM1interaction.

Additionally, we sought to demonstrate that an affinity support/membranemimic could be prepared by pre-incubating an array of artificialreceptors with GM1 which would then bind/capture cholera toxin in abinding pattern which could be used to select a working artificialreceptor(s) for, for example, the high-throughput screen of leadcompounds which will disrupt the “cholera: membrane˜GM1 mimic”. The GM1pre-incubation studies clearly demonstrated that several of the workingartificial receptors which were poor cholera binders significantlyincreased their cholera binding, presumably through an affinityinteraction between the cholera toxin and BOTH the immobilized GM1pentasaccharide moiety and the working artificial receptor buildingblock environment.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

It should also be noted that, as used in this specification and theappended claims, the phrase “adapted and configured” describes a system,apparatus, or other structure that is constructed or configured toperform a particular task or adopt a particular configuration. Thephrase “adapted and configured” can be used interchangeably with othersimilar phrases such as arranged and configured, constructed andarranged, adapted, constructed, manufactured and arranged, and the like.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All of the U.S. patents and published U.S. patentapplications referenced in this application are incorporated byreference as if fully reproduced herein.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. An electrochemical sensing system comprising: a working electrode; areference electrode; and a working artificial receptor that is coupledto the working electrode, wherein the sensing system is configured togenerate a sensing signal.
 2. The electrochemical sensing system ofclaim 1 further comprising a membrane between the working electrode andthe reference electrode, the working artificial receptor being coupledto the membrane.
 3. The electrochemical sensing system of claim 1wherein the system is configured to detect a chemical reaction bydetecting a flow of electrons between the working electrode and thereference electrode.
 4. The electrochemical sensing system of claim 1wherein the system is configured to detect a chemical reaction bydetecting a potential difference between the reference electrode and theworking electrode.
 5. The electrochemical sensing system of claim 1comprising an array of working electrodes.
 6. The electrochemicalsensing system of claim 1 wherein the working receptors are coupled to acarbon paste that is electrically coupled to the working electrode. 7.The sensing system of claim 1, wherein the system is configured todetect at least one of: pathogenic microorganism, cancerous cell,pollutant in water, airborne pollutant, explosive-related vapor,protein, polynucleotide.
 8. An electrochemical sensing systemcomprising: a field effect transistor; a working artificial receptorthat is coupled to the field effect transistor, wherein a signal can begenerated when a test ligand binds to a working artificial receptor. 9.The electrochemical sensing system of claim 8 wherein the field effecttransistor is one of: a ChemFET, an ISFET, a SAFET, an SGFET.
 10. Theelectrochemical sensing system of claim 8 wherein the working artificialreceptor is coupled to a gate.
 11. The electrochemical sensing system ofclaim 8 wherein the working artificial receptor is coupled to aninsulating layer.